Search PubMed⌕ Search

Biomedical subjects

David T Curiel

Publications and source records attributed to David T Curiel.

At least 37 records · Page 2Linked to original sources
PubMed2006

Core labeling of adenovirus with EGFP.

The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expression vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology.

Adenoviridae↗
PubMed2006

Targeted and shielded adenovectors for cancer therapy.

Conditionally replicative adenovirus (CRAd) vectors are novel vectors with utility as virotherapy agents for alternative cancer therapies. These vectors have already established a broad safety record in humans and overcome some of the limitations of non-replicative adenovirus (Ad) vectors. In addition, one potential problem with these vectors, attainment of tumor or tissue selectivity has widely been addressed. However, two confounding problems limiting efficacy of these drug candidates remains. The paucity of the native Ad receptor on tumor tissues, and host humoral response due to pre-existing titers of neutralizing antibodies against the vector itself in humans have been highlighted in the clinical context. The well-characterized CRAd, AdDelta24-RGD, is infectivity enhanced, thus overcoming the lack of coxsackievirus and adenovirus receptor (CAR), and this agent is already rapidly progressing towards clinical translation. However, the perceived host humoral response potentially will limit gains seen from the infectivity enhancement and therefore a strategy to blunt immunity against the vector is required. On the basis of this caveat a novel strategy, termed shielding, has been developed in which the genetic modification of a virion capsid protein would provide uniformly shielded Ad vectors. The identification of the pIX capsid protein as an ideal locale for genetic incorporation of shielding ligands to conceal the Ad vector from pre-existing neutralizing antibodies is a major progression in the development of shielded CRAds. Preliminary data utilizing an Ad vector with HSV-TK fused to the pIX protein indicates that a shield against neutralizing antibodies can be achieved. The utility of various proteins as shielding molecules is currently being addressed. The creation of AdDelta24S-RGD, an infectivity enhanced and shielded Ad vector will provide the next step in the development of clinically and commercially feasible CRAds that can be dosed multiple times for maximum effectiveness in the fight against cancers in humans.

Adenoviridae↗
PubMed2006

Triple-targeted oncolytic adenoviruses featuring the cox2 promoter, E1A transcomplementation, and serotype chimerism for enhanced selectivity for ovarian cancer cells.

Conditionally replicating adenoviruses (CRAd's) feature selective replication in and killing of tumor cells. Initial clinical studies with relatively attenuated early generation agents have resulted in promising safety and efficacy data. Nevertheless, increased specificity may be advantageous for an emerging generation of infectivity-enhanced CRAd's. Further, increased specificity could translate into a larger tolerated dose. An approach for increasing specificity is dual control of E1A expression. We constructed six CRAd's featuring two variants of the cyclo-oxygenase 2 (cox2) promoter, combined with three versions of E1A. Transcriptional targeting was supplemented with transductional targeting utilizing the serotype 3 knob. In vivo and in vitro results suggest that cox2 can be utilized for enhancing the specificity of E1A deletion mutants and that combination with the Delta24 mutation increases specificity without reducing potency. Combination with Delta2-Delta24 was specific but somewhat attenuated. The promoter variants behaved similarly, although the longer 1,554-bp version displayed a trend for improved specificity. Transcriptional modifications were compatible with transductional targeting and resulted in up to 100,000-fold increase in the therapeutic window for Ad5/3cox2Ld24 vs wild-type adenovirus. Thus, the proposed triple-targeting strategy may be useful for increasing the safety and efficacy of adenoviral gene therapy for ovarian cancer.

Adenoviridae↗
PubMed2006

An adenovirus serotype 5 vector with fibers derived from ovine atadenovirus demonstrates CAR-independent tropism and unique biodistribution in mice.

Many clinically important tissues are refractory to adenovirus (Ad) infection due to negligible levels of the primary Ad5 receptor the coxsackie and adenovirus receptor CAR. Thus, development of novel CAR-independent Ad vectors should lead to therapeutic gain. Ovine atadenovirus type 7, the prototype member of genus Atadenovirus, efficiently transduces CAR-deficient human cells in vitro, and systemic administration of OAdV is not associated with liver sequestration in mice. The penton base of OAdV7 does not contain an RGD motif, implicating the long-shafted fiber molecule as a major structural dictate of OAdV tropism. We hypothesized that replacement of the Ad5 fiber with the OAdV7 fiber would result in an Ad5 vector with CAR-independent tropism in vitro and liver "detargeting" in vivo. An Ad5 vector displaying the OAdV7 fiber was constructed (Ad5Luc1-OvF) and displayed CAR-independent, enhanced transduction of CAR-deficient human cells. When administered systemically to C57BL/6 mice, Ad5Luc1-OvF reporter gene expression was reduced by 80% in the liver compared to Ad5 and exhibited 50-fold higher gene expression in the kidney than the control vector. To our knowledge, this is the first report of a fiber-pseudotyped Ad vector that simultaneously displays decreased liver uptake and a distinct organ tropism in vivo. This vector may have future utility in murine models of renal disease.

Animals↗
PubMed2006

mda-7/IL-24: multifunctional cancer-specific apoptosis-inducing cytokine.

"Differentiation therapy" provides a unique and potentially effective, less toxic treatment paradigm for cancer. Moreover, combining "differentiation therapy" with molecular approaches presents an unparalleled opportunity to identify and clone genes mediating cancer growth control, differentiation, senescence, and programmed cell death (apoptosis). Subtraction hybridization applied to human melanoma cells induced to terminally differentiate by treatment with fibroblast interferon (IFN-beta) plus mezerein (MEZ) permitted cloning of melanoma differentiation associated (mda) genes. Founded on its novel properties, one particular mda gene, mda-7, now classified as a member of the interleukin (IL)-10 gene family (IL-24) because of conserved structure, chromosomal location, and cytokine-like properties has become the focus of attention of multiple laboratories. When administered by transfection or adenovirus-transduction into a spectrum of tumor cell types, melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) induces apoptosis, whereas no toxicity is apparent in normal cells. mda-7/IL-24 displays potent "bystander antitumor" activity and also has the capacity to enhance radiation lethality, to induce immune-regulatory activities, and to inhibit tumor angiogenesis. Based on these remarkable attributes and effective antitumor therapy in animal models, this cytokine has taken the important step of entering the clinic. In a Phase I clinical trial, intratumoral injections of adenovirus-administered mda-7/IL-24 (Ad.mda-7) was safe, elicited tumor-regulatory and immune-activating processes, and provided clinically significant activity. This review highlights our current understanding of the diverse activities and properties of this novel cytokine, with potential to become a prominent gene therapy for cancer.

Adenoviridae↗
PubMed2006

Dynamic monitoring of oncolytic adenovirus in vivo by genetic capsid labeling.

BACKGROUND: Conditionally replicative adenoviruses represent a promising strategy to address the limited efficacy and safety issues associated with conventional cancer treatment. Despite rapid translation into human clinical trials and demonstrated safety, the fundamental properties of oncolytic adenovirus replication and spread and host-vector interactions in vivo have not been completely evaluated. METHODS: We developed a noninvasive dynamic monitoring system to detect adenovirus replication. We constructed capsid-labeled E1/E3-deleted and wild-type adenoviruses (Ad-wt) by fusing the minor capsid protein IX with red fluorescent proteins mRFP1 and tdimer2(12), resulting in Ad-IX-mRFP1, Ad-IX-tdimer2(12), and Ad-wt-IX-mRFP1. Virus DNA replication, encapsidation, cytopathic effect, thermostability, and binding to primary receptor (coxsackie adenovirus receptor) were analyzed using real-time quantitative polymerase chain reaction, cell viability (MTS) assay, and fluorescence microscopy. Athymic mice (n = 4) carrying xenograft tumors that were derived from A549 lung adenocarcinoma cells were intratumorally inoculated with Ad-wt-IX-mRFP1, and adenovirus replication was dynamically monitored with a fluorescence noninvasive imaging system. Correlations between fluorescence signal intensity and viral DNA synthesis and replication were calculated using Pearson's correlation coefficient (r). RESULTS: The red fluorescence label had little effect on viral DNA replication, encapsidation, cytopathic effect, thermostability, and coxsackie adenovirus receptor binding. The fluorescent signal correlated with viral DNA synthesis and infectious progeny production both in vitro and in vivo (in A549 cells, r = .99 and r = .65; in tumors, r = .93 and r = .92, respectively). The replication efficiency of Ad-wt-IX-mRFP1 in vivo was variable, and replication and viral spreading and persistence were limited, consistent with clinical observations. CONCLUSIONS: Genetic capsid labeling provides a promising approach for the dynamic assessment of oncolytic adenovirus function in vivo.

Adenocarcinoma↗
PubMed2006

Delta-24 increases the expression and activity of topoisomerase I and enhances the antiglioma effect of irinotecan.

PURPOSE: In this study, we sought to determine whether Delta-24 could sensitize glioma cells to the topoisomerase I inhibitor irinotecan (CPT-11) and to identify the mechanisms underlying this enhanced anticancer effect. EXPERIMENTAL DESIGN: We used human glioblastoma cell lines for the in vitro studies. The expression of topoisomerase I was determined in Western blot analyses, and topoisomerase I activity was determined by measuring the relaxation of a supercoiled DNA. The cell cycle distribution of cells was determined by flow cytometry analysis of the cellular DNA content. Cell viability was quantified by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Tissue culture infection dose assays were used to quantitate adenovirus replication. For the in vivo studies, athymic mice received intracranial/intratumoral injections of Delta-24 in combination with CPT-11, after which animal survival was monitored. RESULTS: Delta-24 infection caused human glioma cells to accumulate in the S phase and induced the expression and activity of topoisomerase I as shown by Western blot and in vitro enzymatic activity assays. Further, we showed that the sequential administration of Delta-24 and CPT-11 to human glioma cell cultures potentiated the CPT-11-mediated anticancer effect in vitro without modifying the replicative phenotype of the oncolytic adenovirus. In vivo experiments showed that the single intratumoral administration of Delta-24 to intracranially implanted human glioma xenografts followed by the systemic administration of CPT-11 resulted in significantly prolonged animal survival. CONCLUSIONS: The combination of Delta-24 treatment with CPT-11 showed an enhanced anticancer effect, which suggests that the interaction between adenoviral and human proteins can be exploited in rational anticancer therapies comprising replication-competent adenoviruses and conventional chemotherapeutic agents.

Adenoviridae↗
PubMed2006

In vivo imaging of adenovirus transduction and enhanced therapeutic efficacy of combination therapy with conditionally replicating adenovirus and adenovirus-p27.

Gene therapy is hampered by poor gene transfer to the tumor mass. We previously proposed a combination adenoviral gene therapy containing a conditionally replicating adenovirus (CRAD) expressing mutant E1 (delta24RGD) and a replication-defective E1-deleted adenovirus to enhance the efficiency of gene transfer. Mutant E1 expressed by delta24RGD enables the replication of replication-defective adenoviruses in tumors when cancer cells are co-infected with both viruses. In this study, gene transfer rates in xenografts tumors were monitored by bioluminescence in cells infected with the replication-defective adenovirus-luciferase (ad-luc). Tumor masses treated with CRAD + ad-luc showed dramatically stronger and more prolonged luciferase expression than ad-luc-treated tumors and this expression spread through the entire tumor mass without significant systemic spread. Transduction with CRAD + replication-defective adenovirus-p27 increased the expression of p27 by 24-fold versus transduction with ad-p27 alone. Treatment of a lung cancer cell line and of established lung cancer xenografts with CRAD + adenovirus-p27 also induced stronger growth suppression than treatment with either virus alone. These findings confirm the selective replication of E1-deleted adenovirus containing a therapeutic gene due to the presence of mutant E1 produced by delta24RGD in tumors. Moreover, this replication increased the therapeutic gene transfer rate and enhanced its antitumor effects.

Adenoviridae↗
PubMed2006

Ionizing radiation enhances adenoviral vector expressing mda-7/IL-24-mediated apoptosis in human ovarian cancer.

Ovarian cancer is the fifth most common cause of cancer-related death in women. Current interventional approaches, including debulking surgery, chemotherapy, and/or radiation have proven minimally effective in preventing the recurrence and/or mortality associated with this malignancy. Subtraction hybridization applied to terminally differentiating human melanoma cells identified melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), whose unique properties include the ability to selectively induce growth suppression, apoptosis, and radiosensitization in diverse cancer cells, without causing any harmful effects in normal cells. Previously, it has been shown that adenovirus-mediated mda-7/IL-24 therapy (Ad.mda-7) induces apoptosis in ovarian cancer cells, however, the apoptosis induction was relatively low. We now document that apoptosis can be enhanced by treating ovarian cancer cells with ionizing radiation (IR) in combination with Ad.mda-7. Additionally, we demonstrate that mda-7/IL-24 gene delivery, under the control of a minimal promoter region of progression elevated gene-3 (PEG-3), which functions selectively in diverse cancer cells with minimal activity in normal cells, displays a selective radiosensitization effect in ovarian cancer cells. The present studies support the use of IR in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in ovarian cancer, particularly in the context of tumors displaying resistance to radiation therapy.

Adenoviruses, Human↗
PubMed2006

Covalently linked Au nanoparticles to a viral vector: potential for combined photothermal and gene cancer therapy.

Hyperthermia can be produced by near-infrared laser irradiation of gold nanoparticles present in tumors and thus induce tumor cell killing via a bystander effect. To be clinically relevant, however, several problems still need to be resolved. In particular, selective delivery and physical targeting of gold nanoparticles to tumor cells are necessary to improve therapeutic selectivity. Considerable progress has been made with respect to retargeting adenoviral vectors for cancer gene therapy. We therefore hypothesized that covalent coupling of gold nanoparticles to retargeted adenoviral vectors would allow selective delivery of the nanoparticles to tumor cells, thus feasibilizing hyperthermia and gene therapy as a combinatorial therapeutic approach. For this, sulfo-N-hydroxysuccinimide labeled gold nanoparticles were reacted to adenoviral vectors encoding a luciferase reporter gene driven by the cytomegalovirus promoter (AdCMVLuc). We herein demonstrate that covalent coupling could be achieved, while retaining virus infectivity and ability to retarget tumor-associated antigens. These results indicate the possibility of using adenoviral vectors as carriers for gold nanoparticles.

Adenoviridae↗
PubMed2006

In vivo analysis of adenovirus-specific cytotoxic T lymphocyte response in mice deficient in CD28, fas ligand, and perforin.

Adenoviruses (Ad) have been extensively studied as gene delivery vectors in gene therapy and as vaccine carriers. The cell-mediated cytotoxicity induced by Ad is of great interest in both applications. However, the mechanism underlying Ad-specific cytotoxic T lymphocyte (CTL) generation and effector function remains unclear. In this study, we used a novel MHC class I tetramer and an in vivo CTL assay to examine the role of CD28, perforin, Fas ligand (FasL), and TNF-alpha in the generation and function of Ad-specific CTLs in vivo. During the primary response, there was a significant defect in both the generation and in vivo effector function of Ad-specific CTLs in CD28-/- mice, but not in CD4+ T cell-depleted mice or CD4-/- mice. The relative role of CTL effector molecules was assayed by in vivo CTL assay in perforin- or FasL-mutant mice, using donor cells from Fas-deficient or TNFR1/TNFR2-deficient mice. The results indicated that the in vivo CTL activity is mediated mainly by perforin. In the absence of perforin, production of FasL, but not TNF-alpha, by the CTLs results in lower level Ad-specific killing of target cells. These results provide important implications concerning the development of safe and effective Ad vectors for gene therapy and vaccines.

Adenoviridae↗
PubMed2006

Mesenchymal progenitor cells as cellular vehicles for delivery of oncolytic adenoviruses.

Natural and genetically modified oncolytic viruses have been systematically tested as anticancer therapeutics. Among this group, conditionally replicative adenoviruses have been developed for a broad range of tumors with a rapid transition to clinical settings. Unfortunately, clinical trials have shown limited antitumor efficacy partly due to insufficient viral delivery to tumor sites. We investigated the possibility of using mesenchymal progenitor cells (MPC) as virus carriers based on the documented tumor-homing abilities of this cell population. We confirmed preferential tumor homing of MPCs in an animal model of ovarian carcinoma and evaluated the capacity of MPCs to be loaded with oncolytic adenoviruses. We showed that MPCs were efficiently infected with an adenovirus genetically modified for coxsackie and adenovirus receptor-independent infection (Ad5/3), which replicated in the cell carriers. MPCs loaded with Ad5/3 caused total cell killing when cocultured with a cancer cell line. In an animal model of ovarian cancer, MPC-based delivery of the Ad5/3 increased the survival of tumor-bearing mice compared with direct viral injection. Further, tumor imaging confirmed a decrease in tumor burden in animals treated with oncolytic virus delivered by MPC carriers compared with the direct injection of the adenovirus. These data show that MPCs can serve as intermediate carriers for replicative adenoviruses and suggest that the natural homing properties of specific cell types can be used for targeted delivery of these virions.

Adenoviridae↗
PubMed2006

Enhanced transduction of malignant glioma with a double targeted Ad5/3-RGD fiber-modified adenovirus.

Malignant brain tumors remain refractory to adenovirus type 5 (Ad5)-based gene therapy, mostly due to the lack of the primary Ad5 receptor, the coxsackie and adenovirus receptor, on brain tumor cells. To bypass the dependence on coxsackie and adenovirus receptor for adenoviral entry and infectivity, we used a novel, double targeted Ad5 backbone-based vector carrying a chimeric Ad5/3 fiber with integrin-binding RGD motif incorporated in its Ad3 knob domain. We then tested the new virus in vitro and in vivo in the setting of malignant glioma. Ad5/3-RGD showed a 10-fold increase in gene expression in passaged cell lines and up to 75-fold increase in primary tumors obtained from patients relative to the control. These results were further corroborated in our in vivo human glioma xenograft model, where the Ad5/3-RGD vector showed a 1,000-fold increase in infectivity as compared with the control. Taken together, our findings indicate that Ad5/3-RGD may be a superior vector for applications in glioma gene therapy and therefore warrants further attention in the field of neuro-oncology.

Adenoviridae↗
PubMed2006

Current developments in adenovirus-based cancer gene therapy.

Adenovirus (Ad)-based cancer gene therapy is a promising, novel approach for treating cancer resistant to established treatment modalities. Unfortunately, the efficacy of nonreplicative first generation Ads was low and data from clinical trials were disappointing. To address this problem, conditionally replicating Ads have been constructed. Infection of tumor cells with conditionally replicating Ads results in tumor-specific replication, subsequent oncolysis and release of the virus progeny. Recently, it has been suggested that the low expression of the coxsackie-Ad receptor is the rate-limiting factor for infectivity with serotype 5 (Ad5). Unfortunately, coxsackie-Ad receptor expression is highly variable and often low on many tumor types. Consequently, molecular strategies have been applied for the development of coxsackie-Ad receptor-independent oncolytic Ads. This review describes recent developments of Ad-based cancer gene therapy, including novel engineering techniques of the Ad capsid for efficient tumor targeting, as well as targeting techniques, to restrict transgene expression to cancer cells.

Adenoviridae↗
PubMed2006

The human survivin promoter: a novel transcriptional targeting strategy for treatment of glioma.

OBJECT: Malignant brain tumors have been proved to be resistant to standard treatments and therefore require new therapeutic strategies. Survivin, a recently described member of the inhibitor of apoptosis protein family, is overexpressed in several human brain tumors, primarily gliomas, but is downregulated in normal tissues. The authors hypothesized that the expression of tumor-specific survivin could be exploited for treatment of gliomas by targeting the tumors with gene therapy vectors. METHODS: Following confirmation of survivin expression in glioma cell lines, an adenoviral vector containing the survivin promoter and the reporter gene luciferase was tested in established and primary glioma cells, normal astrocytic cells, and normal human brain tissues. High levels of reporter gene expression were observed in established tumor and primary tumor cell lines and low levels of expression in astrocytes and normal human brain tissue. To test oncolytic potency, the authors constructed survivin promoter-based conditionally replicative adenoviruses (CRAds), composed of survivin promoter-regulated E1 gene expression and an RGD-4C capsid modification. These CRAds could efficiently replicate within and kill a variety of established glioma tumor cells, but were inactive in a normal human liver organ culture. Finally, survivin promoter-based CRAds significantly inhibited the growth of glioma xenografts in vivo. CONCLUSIONS: Together these data indicate that the survivin promoter is a promising tumor-specific promoter for transcriptional targeting of adenovirus-based vectors and CRAds for malignant gliomas. The strategy of using survivin-CRAds may thus translate into an experimental therapeutic approach that can be used in human clinical trials.

Adenoviridae↗
PubMed2006

A conditionally replicative adenovirus that codes for a TK-GFP fusion protein (Ad5Delta24TK-GFP) for evaluation of the potency of oncolytic virotherapy combined with molecular chemotherapy.

The utility of conditionally replicative adenoviruses (CRAds) in cancer gene therapy is based on their ability to destroy tumor cells by oncolysis. However, in order to achieve adequate therapeutic response, CRAds have to spread through the tumor tissue by replication. Thus, to study the potency of these viruses to replicate and penetrate in tumors, we created a herpes simplex virus type 1 thymidine kinase-green fluorescent protein fusion gene (TK-GFP) encompassing CRAd (Ad5Delta24TK-GFP), whose tumor selectivity is mediated by a retinoblastoma (Rb)-binding site mutation. In addition, we evaluated the oncolytic efficacy of Ad5Delta24TK-GFP in combination with the TK/ganciclovir (GCV) system. Based on our results, Ad5Delta24TK-GFP replicates in cancer cells resulting in oncolysis and can efficiently penetrate into tumors. Additionally, the combination of GCV with Ad5Delta24TK-GFP augmented cell death in vitro but this was not observed in vivo: tumor growth was significantly reduced by oncolysis when compared to non-replicative virus (p<0.001), but administration of GCV did not significantly enhance oncolysis. This suggests that in certain conditions, TK/GCV-mediated cell killing may be counterproductive to replication and oncolysis, which on the other hand might be useful feature for clinical trials in case of replication-associated toxicity.

Adenoviridae↗