Biomedical subjects

David T Curiel

Publications and source records attributed to David T Curiel.

177 records · Page 10Linked to original sources

PubMed2002

The organotypic multicellular spheroid is a relevant three-dimensional model to study adenovirus replication and penetration in human tumors in vitro.

The use of adenoviruses for gene transfer and as oncolytic agents is currently receiving widespread attention. As specific constraints to adenovirus distribution and spread cannot be studied in cell cultures, there is a need for an in vitro three-dimensional (3D) model mimicking the in vivo biology of tumors. We studied the interactions between tumor and adenoviruses using multicellular spheroids grown from primary brain tumor material. Using beta-galactosidase and luciferase reporter genes expressed by replication-defective adenoviruses, we showed that infection was restricted to the first layer of cells. Using a replication-competent adenovirus expressing the luciferase gene, we showed that transgene expression in the spheroid was considerably enhanced and that viral spreading deep into the 3D structure took place. In addition, a tetrazolium salt-based metabolic assay could be used to compare the oncolytic activity of different concentrations of replication-competent adenoviruses. We can conclude that organotypic spheroids offer a versatile in vitro system for studying distribution, spread, and oncolysis by adenoviruses in a clinically relevant model.

Adenoviridae↗

PubMed2002

The presence of the adenovirus E3 region improves the oncolytic potency of conditionally replicative adenoviruses.

PURPOSE: The initial development of conditionally replicative adenoviruses (CRAds) for cancer treatment has aimed at achieving selective replication in and killing of malignant cells. Other aspects such as the potentiation of the cytolytic capacity have also been investigated but still require new endeavors. As an extension of our prior work, we analyzed the effect of the E3 region, which includes the adenovirus death protein, in the context of CRAd oncolytic potency. EXPERIMENTAL DESIGN: We constructed E3-positive (E3+) and E3-negative (E3-) variants of the previously characterized CRAd, Ad5-Delta 24, and its infectivity enhanced version, Ad5-Delta 24RGD, and compared their oncolytic effect in human cancer cell lines infected with 0.01 viral particle/cell and in s.c. xenografts of A549 human lung cancer cells injected intratumorally with a single dose of 10(7) adenoviral particles in immunodeficient mice. RESULTS: The in vitro experiments showed that the E3+ viruses kill tumor cells 1.6-20 times more effectively in different cell lines. As well, the in vivo study demonstrated that the administration of E3+ CRAds resulted in a more potent oncolytic effect compared with the same dose of their E3- counterparts 35 days after virus administration. Moreover, a time course study of virus replication within the tumor xenografts established a correlation between higher in situ propagation of E3+ CRAds and tumor growth inhibition compared with E3- viruses. CONCLUSIONS: These results indicate that the presence of E3 can enhance the antitumor potency of CRAds over and above the levels conferred by the enhancement of infectivity via Arg-Gly-Asp (RGD).

Adenoviridae↗

PubMed2002

Conditionally replicative adenoviruses for ovarian cancer therapy.

The purpose of this review article is to present a logical rationale for the investigation of conditionally replicative adenoviral vectors for the treatment of ovarian carcinoma. A medline database search was performed to identify relevant articles in the English language for the years 1966 to present. The key words used included replicative adenovirus, conditionally replicative adenovirus, transcriptional targeting, replication selective adenovirus, and "ONYX." A total of 89 references were identified and reviewed. Each reference was reviewed for relevance to clinical translation of conditionally replicative adenoviral vector therapy for ovarian cancer. Data from current clinical trials would suggest that potential obstacles for effective replicative viral therapy of ovarian carcinoma include efficient tumor cell infection, restrictions of the cell surface coxsackie and adenovirus receptor, rapid clearance of vector in the ascites environment, tumor cells specificity, and limitations of current findings of clinical trials. The articles were, therefore, evaluated and included if they addressed these shortcomings. Current data would suggest that advanced generation conditionally replicative adenoviral vectors will soon be available for clinical trials in ovarian cancer. Ovarian cancer, because of expression of targetable receptors, transducible cells, and containment within the i.p. cavity, represents a solid tumor suited uniquely for investigation with advanced generation conditionally replicative adenoviral vectors.

Adenoviridae↗

PubMed2002

A model system for the design of armed replicating adenoviruses using p53 as a candidate transgene.

Cancer gene therapy endeavors to overcome the low therapeutic index of currently available therapeutic modalities via the efficient and safe delivery of genetic material into tumor cells. However, despite promising preclinical results, replication-deficient viral vectors have demonstrated a limited efficacy in the clinical setting. To increase vector efficiency, replication-competent viruses have been proposed. Clinical trials have shown the safety of locally injected, conditionally replicative adenoviruses (Ads) but have underscored the need for improved potency. To further increase the therapeutic effect of replicating viral vectors, armed therapeutic viruses (ATVs) have recently been used for high-efficiency transgene expression. However, interference with cellular signaling and viral production by constitutive transgene expression may be counterproductive for ATV replication, thereby hindering the therapeutic outcome. Consequently, studies are equivocal with regard to the potential benefits of ATVs. To address this issue, we hypothesized that induction of replication of an Ad expressing p53 may be a useful strategy in the context of ATV because p53 does not interfere with Ad replication and may even increase its cytolytic effect. We show that in our in vitro ATV model system, E1 transcomplementation of a replication-deficient Ad encoding p53 resulted in dramatic augmentation of cell killing and circumvented resistance to apoptosis. Correlation was found between the degrees of cell killing and apoptosis induction, rather than with viral burst. Furthermore, both Ad5 E1B 55kDa and E4 orf6 genes were required to enhance the cell killing. In conclusion, our p53-ATV model system demonstrates the potential utility of therapeutic transgene expression by a replicating Ad after a rational selection of a candidate transgene.

Adenoviridae↗

PubMed2000

Blood clearance rates of adenovirus type 5 in mice.

Persistence of adenovirus type 5 in blood has implications for the pathogenicity of the virus infection and for the use of this virus in oncolysis and gene therapy. In this study, the kinetics of adenovirus clearance from blood in mice has been evaluated. After a single inoculation of concentrated virus into the vena cava, virus half-life was less than 2 min. Depletion of Kupffer cells (KC) resulted in increased viraemia. After tail-vein injection, virus and latex beads co-localized within KC. An important factor in clearance by KC is the negative charge of particles. Deletion of the hexon hypervariable region 1 acidic stretch decreased the negative charge of the virion but it did not increase blood persistence. Coating with PEG ('PEGylation') reduced the clearance rate but also reduced infectivity.

Animals↗

PubMedDate not supplied

Noninvasive of adenovirus tumor retargeting in living subjects by a soluble adenovirus receptor-epidermal growth factor (sCAR-EGF) fusion protein.

PURPOSE: To demonstrate that non-invasive bioluminescent imaging can monitor restricted expression from a nonreplicating adenovirus in which the cyclooxygenase-2 (COX-2) promoter drives firefly luciferase. PROCEDURES: Adenovirus in which the COX-2 promoter drives the firefly luciferase imaging gene was injected intratumorally into xenografts that express relatively low and relatively high levels of COX-2. Adenovirus that expresses Renilla Luciferase from the cytomegalovirus early promoter was co-injected, to normalize for injection, leakage, vascularization, etc. COX-2 restricted firefly luciferase and global Renilla Luciferase activities were measured by optical imaging techniques both in vivo and in isolated tissues. RESULTS: Dramatic reduction in hepatic luciferase expression after intravenous viral injection can be imaged non-invasively in living animals. Following intratumoral injection, luciferase levels in tumor xenografts that express differing endogenous COX-2 levels reflect the luciferase levels observed when these cells are infected in cell culture. Essentially no luciferase expression is observed in liver following intratumoral injection. CONCLUSION: Both tissue restricted expression and transcriptional redirection to tumors expressing COX-2 can be imaged non-invasively following injection of Adenovirus expressing firefly luciferase from the COX-2 promoter.

Adenoviridae↗

PubMedDate not supplied

Noninvasive imaging of transcriptionally restricted transgene expression following intratumoral injection of an adenovirus in which the COX-2 promoter drives a reporter gene.

PURPOSE: To demonstrate the efficacy of repeated, non-invasive optical imaging of reporter gene expression in monitoring the ability of bi-specific recombinant molecules (i) to "transductionally untarget" adenovirus from Coxsackie and Adenovirus Receptor (CAR)-dependent infection of normal tissue and (ii) to "transductionally retarget" infection to specific target cells. PROCEDURES: sCAR-EGF is a recombinant, bi-specific molecule containing the soluble portion of CAR fused to Epidermal Growth Factor. The sCAR moiety binds to the virus and blocks CAR-dependent adenovirus infection. The EGF moity binds to cellular EGF receptors. We used non-invasive optical imaging of firefly luciferase to repeatedly monitor, in living animals, the ability of sCAR-EGF (i) to "transductionally untarget" systemically administered Ad.CMVfLuc, an adenovirus that constitutively expresses luciferase, from normal tissues and (ii) to "transductionally redirect" adenovirus infection in mice to xenograft tumors that express elevated epidermal growth factor (EGF) receptor levels. RESULTS: Systemic injection of sCAR-EGF "coated" adenovirus expressing firefly luciferase from the CMV early promoter, reduces expression of the reporter gene in the liver and facilitates expression of the reporter gene in tumor xenografts expressing high levels of the EGF-receptor. CONCLUSION: Both liver "untargeting" and tumor "retargeting" of adenovirus by recombinant sCAR-EGF can be imaged non-invasively using a luciferase reporter gene.

Adenoviridae↗

PubMedDate not supplied

Development of a therapeutic adenoviral vector for cholangiocarcinoma combining tumor-restricted gene expression and infectivity enhancement.

Cholangiocarcinoma is an invasive malignancy that is most often unresectable upon diagnosis and unresponsive to chemotherapy and radiation. While adenoviral gene therapy has shown promise in treating many tumors, systemic toxicity and low tumor transduction efficiency have hampered its application in many gastrointestinal cancers. To overcome these difficulties, we have constructed an adenoviral vector utilizing a tumor-specific promoter (TSP) for selective transgene expression and a vector with an RGD-motif in the fiber-knob region for infectivity enhancement. In seeking a TSP for cholangiocarcinoma, Secretory Leukoprotease Inhibitor, Midkine, Gastrin Releasing Peptide, VEGF, Cox-2M, and Cox-2L promoters were configures in adenoviral vectors, and evaluated in cholangiocarcinoma cells lines (Oz and SkChA-1). Luciferase assays demonstrated that Cox-2 promoters (M and L) showed the highest promoter activity, with Cox-2M appearing slightly stronger than Cox-2L. Infectivity enhanced vectors with RGD-motif in the fiber-knob region were also constructed with the luciferase transgene driven by a CMV control and the Cox-2M and Cox-2L promoters. Subsequent luciferase assays comparing the unmodified vectors to the RGD-modified versions demonstrated higher levels of luciferase activity than the RGD-infected cells. This paradigm was then applied to a therapeutic HSV-TK/GCV model by constructing RGD-enhanced HSV-TK vectors driven by Cox-2M and Cox-2L promoters. In vitro cytocidal effect analysis confirmed that the RGD-modified, cox-2 (M and L) driven vectors showed a stronger cytocidal effect upon gancyclovir administration than the vectors with wild-type fiber. The Cox-2 promoter demonstrates a favorable selectivity profile for cholangiocarcinoma, and RGD-modification further enhances transduction efficiency. This combination has potential to overcome the obstacles to clinical application of adenoviral gene therapy in cholangiocarcinoma.

Adenoviridae↗

PubMedDate not supplied

A single-component CD40-targeted adenovirus vector displays highly efficient transduction and activation of dendritic cells in a human skin substrate system.

Dendritic cell (DC) based tumor vaccination usually involves the administration of ex vivo generated autologous DC. Transduction of DC by viral vectors in vivo has been proposed as a more standardized and easily clinically applicable approach. Previously, we have reported that an Ad5 vector targeted to CD40 via genetic capsid incorporation of CD40L achieves selective transduction of DC in vitro. In the present study, we evaluate the ability of this vector to deliver transgenes in a stringent human substrate system. We report the capacity of this CD40-targeted vector to infect, with high efficiency, cutaneous DC resident in human skin explants, while simultaneously inducing their activation and maturation. This latest generation of single-component, fully targeted vectors should make feasible the clinical testing of in vivo DC-targeted vaccines.

Adenoviridae↗

PubMedDate not supplied

Genetic targeting strategies for adenovirus.

Adenovirus serotype 5 (Ad5) continues to be regarded as a gene delivery vehicle of high utility for a variety of clinical applications. However, targeting of the virus to alternate, non-native receptors has become a mandate for many gene therapy approaches, as inefficient viral transduction of target tissues has proven detrimental to the utility of Ad5. Thus, various targeting strategies have been endeavored to the end of highly specific cellular transduction, including that of genetic manipulation of the viral capsid. Modification of the tropism-determining fiber protein and other capsid locales has allowed vectorologists to develop vectors that are highly superior to the first-generation adenoviruses employed for gene therapy. Herein, the various genetic targeting strategies for Ad5 are reviewed, and the various schemas in which targeted transduction has been achieved with tropism-modified vectors are outlined.

Adenoviridae↗

PubMedDate not supplied

Strategies to enhance transductional efficiency of adenoviral-based gene transfer to primary human fibroblasts and keratinocytes as a platform in dermal wounds.

Genetically modified keratinocytes and fibroblasts are suitable for delivery of therapeutic genes capable of modifying the wound healing process. However, efficient gene delivery is a prerequisite for successful gene therapy of wounds. Whereas adenoviral vectors (Ads) exhibit superior levels of in vivo gene transfer, their transductional efficiency to cells resident within wounds may nonetheless be suboptimal, due to deficiency of the primary adenovirus receptor, coxsackie-adenovirus receptor (CAR). We explored CAR-independent transduction to fibroblasts and keratinocytes using a panel of CAR-independent fiber-modified Ads to determine enhancement of infectivity. These fiber-modified adenoviral vectors included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7). To evaluate whether transduction efficiencies of the fiber-modified adenoviral vectors correlated with the expression of their putative receptors on keratinocytes and fibroblasts, we analyzed the mRNA levels of CAR, alpha upsilon integrin, syndecan-1, and glypican-1 using quantitative polymerase chain reaction. Analysis of luciferase and green fluorescent protein transgene expression showed superior transduction efficiency of Ad5.pK7 in keratinocytes and Ad5.RGD.pK7 in fibroblasts. mRNA expression of alpha upsilon integrin, syndecan-1 and glypican-1 was significantly higher in primary fibroblasts than CAR. In keratinocytes, syndecan-1 expression was significantly higher than all the other receptors tested. Significant infectivity enhancement was achieved in keratinocytes and fibroblasts using fiber-modified adenoviral vectors. These strategies to enhance infectivity may help to achieve higher clinical efficacy of wound gene therapy.

Adenoviridae↗

PubMedDate not supplied

Enhanced adenovirus infection of melanoma cells by fiber-modification: incorporation of RGD peptide or Ad5/3 chimerism.

The incidence of malignant melanoma has been increasing. Unfortunately, advanced melanomas are rarely curable with standard therapy; therefore, new forms of treatment such as gene therapy are needed. The success of gene delivery or oncolysis depends on the nature of the vector. Adenoviral vectors are advantageous for several reasons; however, they are dependent on CAR (coxsackie and adenovirus receptor) which is deficient or heterogeneously expressed on melanoma cells in situ. Correspondingly, transduction of freshly purified melanoma cells has been shown to be minimal or variable. In order to overcome this shortcoming, it is necessary to construct tropism modified adenoviral vectors. With this goal in mind, we generated two tropism modified vectors, Ad5lucRGD which has an RGD motif incorporated into the HI loop of the fiber knob and Ad5/3luc1 which contains the tail and shaft domain of the Ad5 fiber and the knob domain of the Ad3 fiber. Herein we demonstrate that Ad5/3luc1 infects CAR-negative primary melanoma cells 1128 times better than Ad5luc1 and 34 times better than Ad5lucRGD. Furthermore we show that Ad5/3luc1 and Ad5lucRGD infect via a CAR independent route by blocking the CAR receptor. In addition, we show that the infectivity of the cells correlates with the expression of CAR and Ad3 receptors determined by FACS analysis. Therefore, Ad5/3 is very attractive as a potential therapeutic vector for malignant melanoma.

Adenoviruses, Human↗

PubMedDate not supplied

mda-7/IL-24, a novel cancer selective apoptosis inducing cytokine gene: from the laboratory into the clinic.

An obstacle to effective gene-based cancer therapies is the limited number of cancer-specific growth suppressing and apoptosis-inducing genes. Using a differentiation induction subtraction hybridization (DISH) approach with human melanoma cells, melanoma differentiation associated (mda) genes were isolated that display elevated expression as a function of irreversible growth arrest, cancer reversion and terminal differentiation. This screening paradigm resulted in the cloning of mda-7 in the context of terminal differentiation of human melanoma cells. Based on its structure, chromosomal location, sequence homology and cytokine-like properties, mda-7 has now been renamed IL-24 and classified as a member of the expanding IL-10 cytokine gene family. Expression of mda-7/IL-24 inversely correlates with melanoma progression and administration of mda-7/IL-24 by means of a replication incompetent adenovirus, Ad.mda-7, results in growth suppression and apoptosis in melanoma cells as well as in a broad-spectrum of additional cancer cell types. In contrast, Ad.mda-7 does not elicit deleterious effects in normal cells, including those of epithelial, fibroblast, astrocyte, melanocyte or endothelial origin. Based on these distinctive properties and anti-tumor and anti-angiogenic activities in human tumor xenograft animal models, mda-7/IL-24 has now entered the clinical arena. A Phase I/II clinical trial in patients with advanced carcinomas involving intratumoral administration of mda-7/IL-24 [using a replication incompetent adenovirus; ING241 (Ad.mda-7)] has documented that this gene is safe and well tolerated by patients and a single virus injection elicits apoptosis in a majority of the tumor. Current data suggests that mda-7/IL-24 may function as a dual-acting cytokine in which its normal physiological functions may be related to specific aspects of the immune system and over-expression culminates in cancer-specific apoptosis. This review will provide a prospectus of our current understanding of mda-7/IL-24.

Adenoviridae↗

PubMedDate not supplied

Cell cycle arrest and apoptosis induced by SART-1 gene transduction.

UNLABELLED: The biological function of the SART-1 gene product is demonstrated and its potential as a target for cancer gene therapy is discussed. MATERIALS AND METHODS: The SART-1 gene was transduced by a recombinant adenovirus vector and its expression was promoted by a CMV promoter. RESULTS: The transduction efficiency by recombinant adenoviruses in A549 and MCF-7 cells was determined using a vector expressing luciferase, which showed high expression in the cells. Cell count analysis using Trypan-Blue dye exclusion showed that SART-1 gene transduction inhibited cell growth. Flow cytometry analysis suggested that SART-1 gene transduction induced cell cycle arrest followed by apoptosis. Western blot analysis confirmed that the apoptosis pathway was activated by SART-1 gene transduction. CONCLUSION: These results show that SART-1 gene transduction induces cell cycle arrest leading to apoptosis and suggest the possibility of gene therapy against cancer. In addition, SART-1 is known to be a tumor antigen in a range of cancers recognized by T cells, thus a potential strategy would be the combination of suicide gene therapy with immuno-gene therapy.

Adenoviridae↗

PubMedDate not supplied

Genetic incorporation of a herpes simplex virus type 1 thymidine kinase and firefly luciferase fusion into the adenovirus protein IX for functional display on the virion.

An advantage of the adenoviral vector is its molecular flexibility, which allows for vector tropism modifications for the purpose of cell targeting. In addition to targeting ligands, the capacity to incorporate heterologous peptides has allowed capsid incorporation of other functionalities. We have defined the minor capsid protein IX (pIX) as a locus capable of presenting incorporated ligands on the virion surface. Thus, we sought to exploit the possibility of incorporating functional proteins at pIX. In our current study, we sought to expand the potential utility of our capsid labeling strategy by developing simultaneous imaging capacity for dedicated small animal positron emission tomography and bioluminescence imaging on a single adenoviral vector. Therefore, we constructed an adenovirus that incorporates a fusion protein of herpes simplex virus type 1 thymidine kinase and firefly luciferase (Luc) (TK-Luc) into adenovirus capsid pIX. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional TK-Luc as a component of their capsid surface. Most importantly, Ad-pIX-TK-Luc retained dual enzymatic functions in vitro and in vivo. This dual-modality approach will allow dynamic or real-time imaging analysis of adenovirus-based interventions with maximized analytic flexibility and enhanced resolution potential.

Adenoviridae↗