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Dirk M Nettelbeck

Publications and source records attributed to Dirk M Nettelbeck.

16 recordsLinked to original sources
PubMed2005

A fiber-modified, secretory leukoprotease inhibitor promoter-based conditionally replicating adenovirus for treatment of ovarian cancer.

PURPOSE: The use of conditionally replicating adenoviruses (CRAD) is dependent on molecular differences between tumor cells and nontumor cells. Transcriptional targeting of CRAD replication via tumor-specific promoters is an effective way to control replication regulation. Genetic fiber pseudotyping is an approach for circumventing low expression of the primary adenovirus serotype 5 (Ad5) receptor by using the distinct adenovirus serotype 3 (Ad3) receptor for entry into and subsequent killing of ovarian cancer cells. EXPERIMENTAL DESIGN: In this study, we constructed a fiber-modified CRAD containing the secretory leukoprotease inhibitor (SLPI) promoter to control viral replication via the E1A gene (Ad5/3SLPI). To evaluate the liver toxicity of chimeric 5/3 fiber-modified CRADs, we compared Ad5/3SLPI with Ad5/3Cox-2L, a CRAD with E1A under control of the Cox-2 promoter, and Ad5/3Delta24, a CRAD that replicates in cancer cells inactive in the retinoblastoma/p16 pathway by use of an in vivo hepatotoxicity model and by a model system that uses slices of human liver. RESULTS: We show efficient viral replication and oncolysis of Ad5/3SLPI in both multiple ovarian cancer cell lines and primary tumor cell spheroids as well as therapeutic efficacy in an orthotopic mouse model of peritoneal carcinomatosis. Ad5/3SLPI showed significantly decreased liver toxicity compared with other 5/3 fiber-modified control vectors examined. CONCLUSIONS: In summary, Ad5/3SLPI is a promising vector candidate for treating metastatic ovarian cancer and showed robust virus replication, oncolysis, and in vivo therapeutic efficacy. Ad5/3SLPI showed comparatively low liver toxicity and therefore holds potential for patient use in the clinic.

Adenoviridae↗
PubMed2005

Employment of microarray analysis to characterize biologic differences associated with tropism-modified adenoviral vectors: utilization of non-native cellular entry pathways.

In this study, we have applied high-density oligonucleotide microarray technology to characterize biologic changes associated with adenoviral vector-mediated target cell infection. We infected a human melanoma cell line, M21, with the tropism-modified vectors, Ad5lucRGD and Ad5/3luc1. In addition, we infected the M21 cell line with the Ad5luc1, a vector which primarily exploits the coxsackie and adenovirus receptor, as its primary native receptor. We found significant changes in gene expression of 5492 genes induced by Ad5luc1 infection, 2439 genes induced by Ad5/3luc1 infection, and 1251 genes induced by Ad5lucRGD infection, compared to uninfected cells. Among these changes in gene expression, 783 changes were common to Ad5/3luc1 and Ad5luc1 infections, 266 were common to Ad5lucRGD and Ad5luc1 infections, and 185 changes in gene expression were common to Ad5/3luc1 and Ad5lucRGD infections. Interestingly, 89 changes in gene expression were common to all the three groups, suggesting a commonly affected pathway. This analysis represents a unique application of microarray to study vector-related issues. Furthermore, these studies demonstrate the utility of microarray for characterizing the biologic sequelae of host-vector interaction.

Adenoviridae↗
PubMed2004

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.

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 (AdDeltaE1AIL), the early gene E2B (AdDeltaE2BIL), or the late fiber gene (AdDeltafiberIL). These viruses showed distinct kinetics of transgene expression and luciferase activity. Early after infection, luciferase activities were lower for these viruses, especially for AdDeltaE2BIL, 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 (AdDeltaE1AIL) to six (AdDeltafiberIL) orders of magnitude higher than for AdTL, reflecting virus replication and efficient transgene expression. Similar results were obtained in vivo after intratumoral injection of AdDeltaE2BIL, AdDeltafiberIL, and AdTL. AdDeltafiberIL and the parental virus, Ad5-Delta24, resulted in similar cytotoxicity, but AdDeltaE2BIL and AdDeltaE1AIL were slightly attenuated. Disruption of the expression of neighboring viral genes by insertion of the transgene was minimal for AdDeltaE2BIL and AdDeltafiberIL, but substantial for AdDeltaE1AIL. 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.

Adenoviridae↗
PubMed2004

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.

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.

Adenoviridae↗
PubMed2004

Recombinant bispecific antibodies for the targeting of adenoviruses to CEA-expressing tumour cells: a comparative analysis of bacterially expressed single-chain diabody and tandem scFv.

We have generated two distinct recombinant bispecific antibody molecules for the retargeting of adenoviral vectors to CEA-expressing tumour cells. These antibody molecules were produced by combining the antigen-binding sites of a neutralising anti-fibre knob scFv (S11) and an anti-CEA antibody either in a single-chain diabody format (scDb CEA-S11) or a tandem scFv format (taFv CEA-S11). In order to facilitate expression of taFv CEA-S11 in bacteria we selected from a phage display library taFv molecules with an optimised linker that connects the two scFv fragments. ScDb CEA-S11 and taFv CEA-S11 were expressed and purified in soluble form from the bacterial periplasm with yields of approximately 100 micro g per litre of bacterial culture. In vitro, both bispecific molecules mediated selective and enhanced transduction of CEA-expressing tumour cells by recombinant adenoviruses. These assays did not reveal any differences in efficiency of adenoviral transduction by the two antibody formats. However, compared with taFv CEA-S11, scDb CEA-S11 exhibited a 2- to 3-fold increased stability in human plasma at 37 degrees C. In summary, we could demonstrate that both formats are suitable for adenovirus targeting to tumour cells with similar biological activity in vitro.

Adenoviridae↗
PubMed2004

Evaluation of tissue-specific promoters in carcinomas of the cervix uteri.

BACKGROUND: Gene therapy is a novel approach for treatment of patients with advanced, recurrent, or metastatic cervical cancer. One effective way to direct transgene expression to specific tissues or tumors is the use of tissue-specific-promoters (TSPs). In the context of adenovirus (Ad)-mediated cancer gene therapy it is rational to choose a TSP which is highly expressed in the tumor but has potentially low activity in non-tumor cells, especially the liver. In this study, we have investigated several promoters which fulfill these criteria. Candidate cervical cancer specific TSPs include promoters of the genes for secretory leukoprotease inhibitor (SLPI), cyclooxygenase-2 (COX-2), Midkine (MK), vascular endothelial growth factor receptor type 1 (flt-1), vascular endothelial growth factor (VEGF), Survivin and the receptor for chemokine SDS-1 (CXCR4). METHODS: To evaluate the specific gene expression of the different promoters in the context of cervical cancer, we constructed a panel of E1-deleted Ads that express luciferase under the control of the promoters of interest. We investigated various established cervical cancer cell lines, as well as purified primary cancer cells and normal control cells from the cervix uteri. RESULTS: In all cell lines tested, promoters for MK, VEGF and CXCR4 showed the highest activity. Both MK and VEGF promoters also resulted in a high activity in primary cervical cancer cells. Interestingly, gene expression profiles correlate with luciferase activity in both cell lines and primary cancer samples. CONCLUSIONS: Our study demonstrates that the promoters for MK and VEGF are active in cervical cancer. We believe that both promoters can be successfully employed as TSPs for gene therapy targeted to cervical cancer.

Adenoviridae↗
PubMed2004

Transcriptional targeting of tumors with a novel tumor-specific survivin promoter.

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.

Adenoviridae↗
PubMed2004

Genetic replacement of the adenovirus shaft fiber reduces liver tropism in ovarian cancer gene therapy.

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.

Adenoviruses, Human↗
PubMed2004

Analyses of melanoma-targeted oncolytic adenoviruses with tyrosinase enhancer/promoter-driven E1A, E4, or both in submerged cells and organotypic cultures.

We have generated novel conditionally replicative adenoviruses (CRAds) targeted to melanoma cells. In these adenoviruses, the E4 region (AdDelta24TyrE4) or both E1 and E4 regions (Ad2xTyr) were controlled by a synthetic tyrosinase enhancer/promoter (Tyr2E/P) specific for melanocytes. The properties of these CRAds were compared with wild-type adenovirus (Adwt) and our previous CRAd with a targeted E1A CRII mutation (AdTyrDelta24) in submerged cultures of melanoma cells and nonmelanoma control cells. We showed that AdDelta24TyrE4 had a cell type selectivity similar to AdTyrDelta24 but had a distinct block in viral reproduction in nonmelanoma cells and that Ad2xTyr had an augmented selectivity for melanoma cells. These viruses were additionally tested in organotypic cultures of melanoma cell lines, primary human keratinocytes (PHKs), or mixed cell populations. Unexpectedly, the CRAds exhibited somewhat different cell type selectivity profiles in these cultures relative to those observed in submerged cultures, demonstrating the importance of multiple assay systems. Specifically, AdTyrDelta24 and Ad2xTyr were selective for melanoma cells, whereas AdDelta24TyrE4 exhibited no selectivity, similar to Adwt. AdTyrDelta24 and Ad2xTyr were strongly attenuated in their ability to lyse PHKs in organotypic cultures. Furthermore, Ad2xTyr had a superior melanoma selectivity in organotypic cultures of cocultivated melanoma cells and PHKs. The enhanced selectivity for melanoma cells exhibited by Ad2xTyr provides a window of opportunity for therapeutic application. These studies also demonstrate that organotypic cultures derived from mixtures of tumor and normal cells represent a promising new model for analysis of CRAd specificity and toxicity.

Adenovirus E1A Proteins↗
PubMed2003

Virotherapeutics: conditionally replicative adenoviruses for viral oncolysis.

Viral oncolysis, or virotherapy, is an endeavor to use viruses as therapeutic agents in an effort to exploit their highly evolved qualities of host cell killing and simultaneous multiplication and spread. This review describes the concept of oncolytic adenoviruses, also called conditionally replicative adenoviruses (CRAds), and recent developments--inspired by early clinical results--that aim at the optimization of CRAd efficacy. Molecular strategies applied for the development of oncolytic adenoviruses include (i) the genetic manipulation of the expression and/or function of key regulatory viral proteins in order to restrict viral replication and spread to tumor cells, (ii) the engineering of the adenoviral capsid for efficient and tumor-targeted infection, and (iii) the incorporation of heterologous genes to facilitate combination therapies or tracking of the virus. Initial clinical trials have provided proof-of-concept for adenoviral oncolysis in patients and a favorable safety profile for oncolytic adenoviruses has been demonstrated. In conclusion, adenoviral oncolysis, with its distinct therapeutic mechanism, shows remarkable therapeutic potential. Advanced generations of virotherapeutics are currently in development.

Adenoviridae↗
PubMed2003

An advanced strategy of enhanced specific gene expression for hepatocellular carcinoma.

Alpha-fetoprotein (AFP) is a specific tumor marker for hepatocellular carcinoma (HCC). A gene expression system under AFP promoter/enhancer control would be specific for AFP producing cells, but its low expression level is a problem which must be overcome. For the purpose of AFP promoter enhancement, we constructed two recombinant adenoviral vectors; one containing the AFP promoter domain and transcriptional activator VP16LexA, and another the transcriptional activator binding site, the AFP promoter and the Cre gene. The lacZ gene was transduced and expression of beta-galactosidase was estimated in vitro. We achieved a 3-fold enhancement of gene expression compared with previous transfection of the transcriptional activator gene into AFP producing cells, and 57 to 330-fold higher cell type specificity was maintained as compared with an ordinary gene expression system. This AFP-producing cell specific gene transduction, employing our enhanced gene expression method, may contribute to targeting gene therapies with a variety of vectors.

Biomarkers, Tumor↗
PubMed2002

Novel oncolytic adenoviruses targeted to melanoma: specific viral replication and cytolysis by expression of E1A mutants from the tyrosinase enhancer/promoter.

Malignant melanoma is characterized by growing incidence, early metastasis, and a lack of effective treatment for advanced disease, suggesting a pressing need for novel therapeutic approaches. Conditionally replicative adenoviruses (CRAds) constitute a new and promising strategy for cancer treatment that has been rapidly translated into clinical trials. We engineered novel melanoma-targeted CRAds, AdTyrdelta24 and AdTyrdelta2delta24, by replacing the adenoviral E1A promoter with a cassette containing a polyA sequence and a human tyrosinase enhancer/promoter construct (hTyr2E/P). The small size of this cassette allows retention of the E3 region within these CRAds, which was shown to enhance viral spread and oncolysis. In addition, we introduced mutations (delta24 and delta2delta24) into the viral E1A gene, which attenuate adenoviral replication in quiescent cells. The cell cycle pathways mediating this attenuation are defective in melanoma cells. By analysis of E1A expression, we prove fidelity of hTyr2E/P in the adenoviral genome and in the context of viral replication when an upstream polyA was included. We further show efficient cytotoxicity of AdTyrdelta24 and AdTyrdelta2delta24 in melanoma cell lines and a 100-1000-fold attenuation in cell lines derived from various nonmelanocytic tissues. Virus replication and progeny production of these viruses were similarly selective, resulting in 200-800-fold higher virus yields in melanoma cells versus control cells, thus establishing viral cytolysis and spread as the cause of the observed cell killing. Cytotoxicity of AdTyrdelta24 for normal fibroblasts and keratinocytes was strongly attenuated, and this virus caused selective killing of melanoma cells but not surrounding keratinocytes in a coculture system. Progeny production and cytotoxicity of AdTyrdelta24 in melanoma cells were similar to matching viruses containing the stronger cytomegalovirus enhancer/promoter instead of hTyr2E/P. Furthermore, AdTyrdelta24 showed a cytopathic effect similar to the wild-type E1A containing AdTyrwt and only minimally reduced compared with wild-type adenovirus. We conclude that the generated CRAds AdTyrdelta24 and AdTyrdelta2delta24 constitute novel targeted agents for gene therapy and viral oncolysis of metastatic melanoma.

Adenovirus E1A Proteins↗
PubMed2002

Treatment of ovarian cancer with a tropism modified oncolytic adenovirus.

Ad5-Delta 24RGD is an adenovirus that is selectively replication competent in cells defective in the Rb/p16 pathway, such as ovarian cancer cells. The fiber of Ad5-Delta 24RGD contains an integrin binding RGD-4C motif, allowing Coxsackie adenovirus receptor-independent infection of cancer cells. Oncolysis of cell lines was similar to that of a wild-type control, and replication in primary tumor material was shown using a novel three-dimensional spheroid model. Finally, an orthotopic murine model of peritoneally disseminated ovarian cancer was used to test i.p. administration to tumor-bearing animals. Injection of the agent resulted in eradication of i.p. disease, whereas control animals expired (P < 0.0001). These results suggest that Ad5-Delta 24RGD could be useful for treatment of ovarian cancer in humans.

Adenoviridae↗
PubMed2002

The flt-1 promoter for transcriptional targeting of teratocarcinoma.

Flt-1, a receptor for vascular endothelial growth factor, is known to display dysregulated expression in both tumor vasculature and tumor cells per se, suggesting that the flt-1 promoter might be a useful candidate to achieve tumor-specific transgene expression. In addition, adenoviral vectors containing transgenes under the control of the flt-1 promoter achieve very low levels of expression in the normal liver, the major organ responsible for blood clearance of adenoviruses and inadvertent transgene-related toxicity. Thus, we assessed the ability of adenoviral vectors containing the flt-1 promoter to achieve transgene expression in a range of gynecological and breast tumor lines. High transgene expression levels were detected in teratocarcinoma lines, correlating with levels of flt-1 mRNA. These results suggest that the flt-1 promoter could be useful for transcriptionally targeted gene expression to teratocarcinoma, and that evaluation in other flt-1-positive tumors is warranted.

Animals↗
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↗