Genetic cytoprotection of human endothelial cells during preservation time with an adenoviral vector encoding the anti-apoptotic human Bcl-2 gene.
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Adenoviridae↗
Biomedical subjects
D T Curiel
Publications and source records attributed to D T Curiel.
Combined cytosine deaminase expression, 5-fluorocytosine exposure, and radiotherapy increases cytotoxicity to cholangiocarcinoma cells.
Cholangiocarcinoma is a malignancy that is resistant to current therapy. We applied the toxin gene therapy strategy of cytosine deaminase conversion of the nontoxic producing 5-fluorocytosine to 5-fluorouracil combined with radiotherapy to cholangiocarcinoma. The transduction efficiency of SK-ChA-1 cholangiocarcinoma cells was determined by fluorescence-activated cell-sorting analysis following infection with recombinant adenovirus AdCMVLacZ, which encodes thc gene for Beta-galactosidase. To evaluate cytosine deaminase-mediated conversion of 5-fluorocytosine to 5-fluorouracil and subsequent cytotoxicity, SK-ChA-1 cells were infected with the recombinant adenovirus AdCMVCD, which encodes cytosine deaminase, and exposed to 5-fluorocytosine for 6 to 8 days. Additive cytotoxicity of radiation therapy was evaluated by cobalt-60 exposure following AdCMVCD infection and 5-fluorocytosine treatment. SK-ChA-1 cells were transduced (98.4%) by AdCMVLacZ at 100 plaque-forming units per cell. Following infection with AdCMVCD and exposure to 5 to 100 microgram/ml of 5-fluorocytosine, 20% to 64% of SK-ChA-1 cells were killed. A combination of radiation and cytosine deaminase/5-fluorocytosine therapy resulted in enhanced cell killing (83.5% to 91.5%). Cholangiocarcinoma cells were transduced by recombinant adenoviral vectors and were killed by cytosine deaminase-mediated production of 5-fluorouracil. Enhanced cytotoxicity was seen with the addition of external beam radiation. These results provide a foundation for multimodality therapy for human cholangiocarcinoma that combines gene therapy technology with radiation therapy.
Adenoviridae↗
Specific membrane receptor gene expression targeted with radiolabeled peptide employing the erbB-2 and DF3 promoter elements in adenoviral vectors.
Radioimmunotherapy is limited by a variety of factors, including poor tumor penetration of monoclonal antibodies and low levels of intratumoral antigen expression. To address these limitations, a gene therapy strategy was devised to genetically induce tumor cells to express enhanced levels of membrane receptors with high affinity for a radiolabeled peptide. We designated this approach as genetic radioisotope targeting strategy. To this end, an adenoviral vector (AdCMVGRPr) encoding the murine gastrin-releasing peptide receptor (GRPr) was used to achieve a high level of binding of radiolabeled bombesin (BBN). To achieve genetic induction of membrane GRPr specifically to tumor cells, we constructed two adenoviral vectors encoding the GRPr gene under the control of the tumor-specific regulatory elements, DF3 (AdDF3GRPr) or erbB-2 (AderbGRPr). We investigated the binding of [125I]BBN to the GRPr following infection with AdDF3GRPr and AderbGRPr in a panel of human breast, pancreatic, and cholangiocarcinoma tumor cell lines. [125I]BBN binding and GRPr expression increased with increasing multiplicities of infection of AdCMVGRPr in all of the cell lines tested. Breast cancer cell lines expressing erbB-2 showed significant GRPr expression using AderbGRPr. A similar result was observed in breast and cholangiocarcinoma cells infected with AdDF3GRPr expressing MUC1 as detected by immunohistochemistry but was not seen in the pancreatic cell lines tested. Thus, adenoviral vectors with tissue-specific promoter elements can be used to achieve a selective expression of membrane receptors that can be targeted with a radiolabeled peptide. The use of such a transcriptional targeting approach may restrict gene expression to tumors and limit the radiation dose deposited in normal tissues in vivo.
Adenoviridae↗
[Viral therapy of cancer].
In spite of the advances achieved in cancer therapeutics, conventional therapies still face problems such as toxicity to normal tissues and low efficacy in advanced phases of the disease. One of the most recent options, albeit experimental, is gene therapy. This strategy shows higher specificity than conventional therapies, but the low efficiency of gene transduction is a hurdle yet to be overcome, and the clinical trials have not shown the expected efficacy. Recently, oncolytic viruses have revived as an alternative in the field of cancer therapeutics. By virtue of the advances in virology and molecular biology techniques, the manipulation of viral genomes to attenuate their pathogenicity and the modification of their life cycle to allow tumor specific viral replication are now possible. At the moment, adenovirus and herpes simplex type 1 mutants are the most commonly employed viruses. Recent clinical trials using these mutant viruses have shown safety and efficacy. Moreover, viral therapy has been combined with radiotherapy or chemotherapy demonstrating additive antineoplastic effects. In this manuscript, we review the biology behind this therapy, the obstacles, and the ongoing clinical trials.
Animals↗
High-efficiency gene transfer employing adenovirus-polylysine-DNA complexes.
Gene transfer may be accomplished via the receptor-mediated endocytosis pathway employing molecular conjugate vectors. These synthetic agents consist of two covalently linked functional components: a DNA-binding domain and a domain that is a ligand for a cell surface receptor. Recognition of the ligand by its appropriate cell surface receptor allows cellular internalization of the complex via receptor-mediated endocytosis. Since conjugates are limited because they lack a specific mechanism to accomplish cell vesicle escape, and since adenoviruses possess such a mechanism, we derived conjugates that incorporate adenovirus into their functional design. The adenovirus-polylysine conjugates mediated high-efficiency gene transfer. In this configuration, the viral moiety functions both as an endosomolysis agent and also as the ligand domain of the conjugate. To determine whether the adenovirus component of the conjugate could function exclusively in the former capacity, combination conjugates were constructed containing an alternative ligand in conjugation with an incorporated adenovirus. Internalization of these conjugates via the receptor for the alternative ligand in cells lacking adenovirus receptors demonstrated that the adenovirus moiety could function to enhance conjugate entry in this context. Thus, it is possible to design highly efficient molecular conjugate vectors that possess specific and distinct mechanisms to accomplish cellular internalization and cell vesicle escape.
Adenoviridae↗
Intracellular antibody against erbB-2 mediates targeted tumor cell eradication by apoptosis.
Methods were developed to achieve targeted eradication of the erbB-2 oncoprotein using gene constructs encoding anti-erbB-2 intracellular single-chain antibodies. This method of genetic intervention caused a marked cytocidal effect in erbB-2-overexpressing human ovarian tumor cells. Evaluation of the mechanistic basis of this phenomenon demonstrated that programmed cell death had been induced. Significantly, no cytocidal effect was observed in non-erbB-2-overexpressing tumors. The induction of apoptosis could be shown to be secondary to the intracellular antibody-mediated ectopic localization of the erbB-2 oncoprotein. Thus, the strategy of selective oncogene "knock-out" using intracellular antibodies represents a novel anticancer gene therapy strategy that offers the potential to achieve highly specific, targeted eradication of human tumor cells.
Antibodies↗
Strategies to accomplish gene delivery via the receptor-mediated endocytosis pathway.
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Animals↗
Lipid- and adenoviral-mediated gene transfer into AIDS-Kaposi's sarcoma cell lines.
Kaposi's sarcoma (KS) is the most frequent malignancy occurring in HIV-positive individuals. AIDS-KS is a more aggressive disease than the classical form, frequently having a rapid clinical course with numerous serious complications. Current systemic treatments for KS, such as chemotherapy and the administration of biological modifiers, are complicated by both the drug resistance of the tumor and the dose-limiting toxicity of the reagents. The relative accessibility of many KS lesions makes the disease a particularly attractive candidate for in vivo gene therapy protocols. In this regard, we are interested in delivering conditionally toxic suicide and/or antiangiogenic vectors to accomplish targeted cell death selectively in AIDS-KS cells. To this end, we examined both cationic lipid- and adenoviral-mediated DNA transfection methods. Using the firefly luciferase reporter gene, we optimized numerous variables known to be important in lipid-mediated DNA transfection, including lipid formulation, the amount of lipid and DNA, lipid/DNA ratio, and cell concentration. Under optimal transfection conditions, approximately 5-25% of KS cells expressed the introduced DNA sequences. Adenoviral-mediated DNA delivery was more efficient than lipid delivery in 4 of 5 primary KS cell lines. Two of the lines (RW248 and RW376) were transduced by adenovirus at frequencies approaching 100%; two cell lines (CVU-1 and RW80) gave efficiencies of 20-35%. Two immortalized KS cell lines (KS Y-1 and KS SLK) were poorly infected, giving a transduction efficiency of <5%. These findings demonstrate that gene transfer into AIDS-KS cells is feasible, and suggest that vector strategies may be permissive for translating gene therapy approaches for the disease.
AIDS-Related Opportunistic Infections↗
Use of a tissue-specific promoter for targeted expression of the herpes simplex virus thymidine kinase gene in cervical carcinoma cells.
Molecular chemotherapy strategies have been developed for a number of epithelial malignancies based on selective delivery and expression of a toxin-encoding gene into the cancer cells. To date, these strategies have not been explored in the context of carcinoma of the cervix, despite the fact that a variety of factors suggest this as an appropriate disease for this gene therapy approach. One limitation in this respect is that appropriate tissue-specific promoters for selective toxin gene expression have not been defined for cervical carcinoma. In this regard, the secretory leukoprotease inhibitor (SLPI) gene has been shown to be constitutively expressed in many epithelial carcinoma cells including the uterine cervix. Thus, we investigated the utility of the SLPI gene as a tissue-specific promoter for regulatory control of the herpes simplex virus thymidine kinase gene for in vitro treatment of cervical carcinoma cells. For this analysis, a gene construct was derived with the herpes simplex virus thymidine kinase gene under regulatory control of the 5' upstream regions of the SLPI gene. Transient transduction of three human cervical carcinoma cell lines with the SLPI-thymidine kinase (TK) construct was followed by treatment with the prodrug ganciclovir. Crystal violet staining was subsequently used to assess cell viability. In this analysis, it was shown that the SLPI-TK construct directed TK-mediated killing in two of three tested cervical cell lines, with the two cell lines being positive for SLPI. In addition, mixing experiments established that cervical carcinoma cells could exhibit a bystander effect which potentially augments the efficacy of molecular chemotherapy approaches. These findings may allow for the development of efficacious, target-specific, toxin gene therapy strategies for cervical carcinoma in human patients.
Carcinoma↗