Towards the use of replicative adenoviral vectors for cancer gene therapy.
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Adenoviruses, Human↗
Biomedical subjects
D T Curiel
Publications and source records attributed to D T Curiel.
Enhancement of radiolabeled antibody binding and tumor localization through adenoviral transduction of the human carcinoembryonic antigen gene.
Conventional radiolabeled antibody targeting utilized in radioimmunotherapy has resulted in limited success clinically due in part to inadequate tumor localization resulting from low expression of human tumor-associated antigens on target cells. We hypothesized that one could improve upon these limitations by genetically inducing tumor cells to express high levels of a new membrane-associated receptor with high affinity for a radioligand. As a preliminary strategy, we induced a human glioma cell line (D54 MG) to express human carcinoembryonic antigen (CEA) in vitro. To accomplish this, we constructed a recombinant adenoviral vector encoding the CEA cDNA inserted downstream of a cytomegalovirus (CMV) promoter (AdCMVCEA). D54 MG cells were transfected with AdCMVCEA or an adenoviral vector encoding lacZ reporter gene as a control (AdCMVlacZ). LS174T human colon cancer cells, known to express CEA constitutively, served as positive controls. Immunofluorescence and immunohistochemistry assays employing unlabeled anti-CEA COL-1 monoclonal antibody demonstrated expression of CEA antigen on the cell surface of transduced D54 MG cells in culture. In addition, assays utilizing 125I-labeled COL-1 indicated high binding to transduced D54 MG cells expressing CEA (4.7 +/- 0.5 x 10(5) COL-1 molecules bound per cell) as compared with minimal binding to nontransduced D54 MG cells. LS174T cells demonstrated only 2.7 +/- 0.5 x 10(6) COL-1 molecules bound per cell. Thus, AdCMVCEA was able to induce levels of cell surface CEA in target cells at a higher level than CEA-overexpressing tumor cells (P < 0.01). The efficacy of transduction of recombinant AdCMVCEA by direct intratumoral injection into D54 MG xenografts was investigated by immunohistochemical analysis, immunofluorescence and by measuring 131I-labeled COL-1 uptake through external scintigraphic imaging and biodistribution studies. Expression of CEA in the tumor xenografts by, and radiolabeled antibody tumor targeting to, AdCMVCEA transduced D54 MG xenografts was comparable to that seen with LS174T xenografts. Results of these studies indicate the potential of adenovirus-mediated delivery of targets to improve radiopharmaceutical tumor localization.
Adenoviruses, Human↗
In vivo gene transfer to the human biliary tract.
The human biliary tract offers an excellent model for gene transfer studies for a variety of diseases localized to the liver. The aim of this study was to determine if a viable liver might be employed to study viral transfection of the human biliary system in order to mimic in vivo human experiments. Using a normal human liver initially procured for transplantation, but subsequently found unsuitable, and with an intact biliary tree, the hepatic vascular supply was accessed for continuous perfusion. The common and left hepatic biliary system was isolated by balloon catheterization. A replication defective adenoviral vector containing the Escherichia coli beta-galactosidase (lac Z) reporter gene (AdCMVLacZ) was injected into the catheter-isolated left and common bile duct lumen. Viral exposure to the right duct system was prevented by ligation. The bile duct segments were excised and prepared for enzymatic (X-gal) staining. Intense staining was observed in the biliary epithelium exposed to the adenoviral vector. No evidence of beta-galactosidase staining was noted in the unexposed biliary mucosa. We report direct transfection of biliary epithelial cells from normal human liver with a recombinant adenovirus. Our data suggest potential therapeutic applications for gene therapy of hepatobiliary disorders.
Adenoviridae↗
Selected strategies to augment polynucleotide immunization.
We sought to amplify the immune response to polynucleotide immunization through co-delivery of complementary DNA (cDNA) encoding a cytokine or co-stimulatory molecule to enhance antigen presentation. In the context of intramuscular immunization, we examined co-delivery of cDNAs for B7-1 and human carcinoembryonic antigen (CEA) within separate plasmids or a dual plasmid with two independent expression cassettes. Intramuscular delivery of the dual expression plasmid produced anti-CEA antibody responses and antitumor effects superior to those generated by plasmid DNA encoding CEA alone. However, co-delivery of cDNAs encoding B7-1 and CEA in the form of two separate plasmids produced no augmentation. The importance of single plasmid delivery suggests the effectiveness of this strategy is contingent upon co-expression of B7-1 and CEA within the same cell. The success of cutaneous polynucleotide immunization by particle bombardment is thought to derive largely from the presence of Langerhans cells within the skin. We hypothesized that co-delivery of plasmid DNA encoding granulocyte-macrophage colony stimulating factor (GM-CSF) by particle bombardment would enhance the antigen presenting capacity of Langerhans cells at the inoculation site similar to its effects in vitro. Augmentation of CEA-specific lymphoblastic transformation and antibody response was observed when plasmid GM-CSF (pGM-CSF) was administered 3 days prior to each dose of plasmid DNA encoding CEA. These strategies for augmentation of immune response to polynucleotide immunization should be applicable to a wide variety of antigenic targets including infectious agents and other tumor-associated antigens.
Animals↗
Approaches to enhance cancer radiotherapy employing gene transfer methods.
This review presents an overview and discussion of the potential synergistic strategies of radiation therapy and gene transfer for treating neoplastic disease. Topics discussed include radiation-inducible promoters coupled to genes which produce proteins that are cytotoxic or enhance radiosensitivity, employment of molecular chemotherapy approaches in conjunction with radiation therapy, and genetic induction of radiosensitization through modification of DNA repair, signal transduction, and cell cycle control genes. Additional topics discussed relate to gene transfer augmentation of radioimmunotherapy of cancer. Specifically, gene transfer methods to genetically induce tumor cells to express enhanced levels of cell surface antigens and receptors to increase radiolabeled antibody and peptide targeting and thus increase their therapeutic effect, selection of radionuclides for therapeutic ligand labeling, and computer simulation of genetic tumor-specific delivery of radiolabeled ligands are proposed.
Animals↗
E1A RNA transcripts amplify adenovirus-mediated tumor reduction.
Previous work by this group has established that E1-defective, recombinant adenoviruses can be replication-enabled by the codelivery of a plasmid encoding the deleted E1 functions, a strategy now designated conditional replication-enablement system for adenovirus (CRESA). In the studies reported here, the original replication-enabling plasmid was replaced by two separate plasmids that encoded the necessary E1A and E1B functions, respectively. An RNA transcript encoding the requisite E1A functions was shown to substitute functionally for the E1A plasmid without significant loss of new adenovirus production in in vitro experiments. No replication competent adenovirus was detectable in the cells treated with the plasmids, or the RNA and plasmid combinations. Subcutaneous human tumor nodules containing a fraction of cells cotransduced with the replication-enabling RNA + DNA and an adenovirus containing a herpes simplex virus thymidine kinase (HSVtk) expression cassette were reduced to a greater extent than control nodules containing the same fraction of cells cotransduced with the virus and an irrelevant plasmid. These experiments show that an E1-defective adenovirus can be conditionally replication-enabled by an RNA transcript encoding the required E1 functions, and that the replication-enablement is sufficient to produce an augmentation of an adenovirus-mediated therapeutic effect in vivo.
Adenoviridae↗
Novel gene therapy strategy to accomplish growth factor modulation induces enhanced tumor cell chemosensitivity.
erbB-2 is a cell surface transmembrane glycoprotein which, when overexpressed, has been shown to be relevant to intrinsic tumor cell chemoresistance. Thus, strategies to down-regulate cell surface erbB-2 have resulted in enhanced tumor cell chemosensitivity. We have recently reported a gene therapy strategy to down-modulate erbB-2 expression using a plasmid construct encoding an intracellular single chain antibody. Therefore, we now demonstrate enhanced chemosensitivity to cis-diamminedichloroplatinum in erbB-2 overexpressing tumor cells and a model system of stable clones using an intracellular single chain antibody. These findings are consistent with the hypothesis that erbB-2 plays a role in tumor cell chemoresistance. In addition, these findings represent a novel gene therapy approach to overcome erbB-2-mediated tumor cell chemoresistance.
Cisplatin↗
Neoplastic reversion accomplished by high efficiency adenoviral-mediated delivery of an anti-ras ribozyme.
Strategies have been developed to abrogate the aberrant expression of dominant oncogenes as a means to accomplish targeted tumor eradication. We have demonstrated previously the utility of this approach using a hammerhead ribozyme designed to cleave the mutant sequence in codon 12 of the activated H-ras oncogene transcript. To develop this strategy into a practical means to approach malignant disease, methods must be developed to accomplish high efficiency delivery of the ribozyme to target neoplastic cells. To accomplish this, a recombinant adenovirus was designed that encoded a gene cassette for the H-ras ribozyme. By using this virus, it was possible to accomplish high efficiency reversion of the neoplastic phenotype in mutant H-ras expressing tumor cells without the need for any selection steps. The demonstration of the utility of adenoviral-mediated delivery of anticancer ribozymes will allow the practical development of gene therapy strategies on this basis.
Adenoviridae↗
A Sindbis virus mRNA polynucleotide vector achieves prolonged and high level heterologous gene expression in vivo.
The direct intramuscular delivery of naked plasmid DNA has been demonstrated to allow expression of encoded heterologous genes in the target myocytes. The method has been employed to elicit immunization based upon delivery of antigen encoding plasmid DNA. For application in the context of achieving anti-tumor immunization against antigenic transforming oncoproteins, delivery of plasmid DNAs encoding these molecules would create significant potential safety hazards. As an alternative to DNA polynucleotide vectors, we explored the utility of mRNA vehicles for inducing foreign gene expression in muscle cells in vivo. Synthetic reporter-gene encoding mRNA transcripts were derived for this analysis. The Sindbis virus vector was also used to derive luciferase mRNA transcripts which possessed self-replication capacity. In these studies, it could be shown that the replicative vector was capable of directing significantly elevated levels of reporter gene expression in myocytes compared to a non-replicative mRNA species. In addition, the replicative species was capable of achieving significantly prolonged levels of in vivo gene expression compared to non-replicative mRNA. Both of these characteristics will make replicative mRNA vectors of utility for polynucleotide-based immunization protocols.
Animals↗
Characterization of a messenger RNA polynucleotide vaccine vector.
We have constructed mRNA transcripts encoding luciferase and human carcinoembryonic antigen (CEA) which are capped, polyadenylated, and stabilized by human beta-globin 5' and 3' untranslated regions. The mRNA construct encoding human CEA directed CEA expression in mouse fibroblasts in vitro following liposome-mediated transfection. The luciferase encoding mRNA transcripts mediated luciferase expression in vivo following i.m. injection. Based on the demonstration of protein expression in vitro and in vivo, the feasibility of using such a vector as a tumor vaccine was examined. In this pilot study, seven mice received 50 micrograms mRNA transcripts encoding CEA twice weekly for 5 weeks by i.m. injection followed by challenge with syngeneic, CEA-expressing tumor cells. This dose and schedule "primed" an immune response to CEA. Five of seven mRNA-immunized mice demonstrated anti-CEA antibody 3 weeks after tumor challenge whereas control mice had no evidence of antibody response. This strategy might be particularly useful to induce an immune response to a proto-oncogene product or growth factor which poses a risk of inducing malignant transformation consequent to prolonged protein expression.
Animals↗
Targeted eradication of ovarian cancer mediated by intracellular expression of anti-erbB-2 single-chain antibody.
OBJECTIVE: Overexpression of the tyrosine kinase receptor erbB-2 is important in the pathogenesis of a variety of neoplasms including ovarian cancer. As a strategy to selectively eradicate erbB-2-overexpressing tumor cells, an anti-erbB-2 single-chain immunoglobin (sFv) gene was constructed to direct expression of intracellular anti-erbB-2 antibody. The purpose of this study is to establish the antitumorigenicity of this strategy in in vitro and in vivo models. METHODS: An anti-erbB-2 sFv construct containing an endoplasmic reticulum (ER)-directed leader sequence was transiently expressed in the human ovarian carcinoma cell line SKOV3 using the adenovirus-polylysine vector. SKOV3 cells transfected with a non-ER form of an anti-erbB-2 sFv construct or an irrelevant plasmid DNA served as controls. Antitumorigenicity, as measured by anchorage-independent growth in soft agar and subcutaneous (sc) tumor formation, was analyzed. The ability to achieve a biological effect with relevant sFv in murine orthotopic xenograft models and in primary human ovarian cancer cells was also evaluated. RESULTS: The ER form of anti-erbB-2 sFv was found to exert a marked antineoplastic effect on the SKOV3 cell line resulting in an arrest of anchorage-independent growth. A significant increase in sc tumor volume was noted in animals challenged with control constructs. In marked contrast, complete tumor eradication was noted at necropsy 80 days after sc transplantation in the group challenged with the ER-directed anti-erbB-2 sFv gene. Intraperitoneal treatment of malignant ascites in human tumor xenograft models with the ER form of the anti-erbB-2 sFv gene resulted in profound downregulation of cell surface erbB-2 in retrieved ovarian cancer cells. The ER-directed anti-erbB-2 sFv also elicited a significant cytotoxic effect in transfected primary ovarian cancer cells obtained from a patient with malignant ascites. CONCLUSION: The ability to selectively "knock out" erbB-2 demonstrates that this strategy can induce a significant antineoplastic effect in ovarian cancer cells overexpressing this growth factor receptor. In addition, the ability to accomplish selective abrogation of erbB-2 expression in animal treatment models and to transfect and eradicate primary ovarian cancer cells justifies further investigation of this novel strategy in ovarian cancer patients.
Animals↗
Targeted tumor killing via an intracellular antibody against erbB-2.
Specific killing of erbB-2-overexpressing tumor cells can be achieved using expression of an intracellular antibody directed against the erbB-2 oncoprotein. We have developed a strategy using a recombinant adenovirus encoding an anti-erbB-2 single chain antibody to achieve targeted tumor cell killing in vivo and can show significantly prolonged survival of animals carrying a human ovarian carcinoma tumor burden within their peritoneal cavities. This strategy of gene therapy for ovarian carcinoma offers the potential to achieve highly specific, targeted killing of human tumor cells and thus establishes the rationale to undertake human clinical trials on this basis.
Adenoviridae↗
Complementation of a human adenovirus early region 4 deletion mutant in 293 cells using adenovirus-polylysine-DNA complexes.
The E1 deleted adenoviral vectors are efficient at gene transfer to cells in culture or in animals. However, their use is limited because of an immune-mediated loss of transduced cells. This immune response is believed to result from low-level production of viral antigens from these vectors after gene transfer. The early region 4 (E4) of adenovirus produces a number of proteins that play an important role in adenoviral and host gene regulation during infection of mammalian cells. There is interest in developing E4 deficient adenovirus for gene therapy, especially in the context of developing a combined E1/E4 deleted vector. Towards this goal, a method by which to complement and propagate an E4 deficient adenovirus (dl 1014) in the E1 complementing 293 cell line, using a novel and simple rescue technique, has been developed. Purified adenovirus deficient in E4 gene expression (dl 1014) was conjugated to expression plasmids containing the E4-open reading frame 6 gene or complete E4 region to produce adenovirus-polylysine-DNA complexes that were used to transfect 293 cells. The derived virus obtained from this transfection did not replicate on 293 cells but did replicate on W162 cells (E4+) confirming that the virus was indeed deleted for E4. Viral yield was high ranging from 3 x 10(8) to 9 x 10(8) plaque forming units per 10(6) 293 cells. This method has general application to the production of new adenoviral mutants that will be useful for developing second generation adenoviral vectors.
Adenovirus E4 Proteins↗
A carcinoembryonic antigen polynucleotide vaccine for human clinical use.
We have constructed a plasmid DNA encoding the full-length complementary DNA for human carcinoembryonic antigen (CEA) under transcriptional regulatory control of the cytomegalovirus early promoter/enhancer (pCEA) and demonstrated that this plasmid can function as a polynucleotide vaccine to elicit a CEA-specific immune response. This immune response protects against tumor challenge with syngeneic CEA-transduced colon carcinoma cells in mice. In the present work, the pCEA construct and purification method were modified to eliminate nonessential viral sequences, the ampicillin selectable marker, mutagens, and endotoxin to produce a reagent suitable for human clinical trials. The human use plasmid (pGT37) directs CEA expression at levels comparable with the original pCEA plasmid and can be propagated to yield large quantities of plasmid DNA based on kanamycin selection. A simple extraction technique greatly reduces contamination by endotoxin. Six weekly intramuscular injections of pGT37 elicited CEA-specific lymphoblastic transformation and antibody response in five of five mice and fully protected 10 of 10 mice against tumor challenge with syngeneic CEA-expressing colon cancer cells 42 days from the first plasmid injection. Thus, pGT37 encoding a tumor-associated antigen (CEA) has been shown to elicit cellular and humoral immune responses and mediate antitumor effects in vivo. This plasmid is suitable for human use and can be easily propagated in the laboratory.
Adenocarcinoma↗
Recombinant adenovirus-mediated gene transfer to genitourinary epithelium in vitro and in vivo.
Transitional cell carcinoma (TCC) of the bladder is associated with characterized lesions in dominant and recessive oncogenes. The understanding of the molecular basis of tumorigenesis in these instances makes possible the application of gene therapy strategies for TCC. In this regard, the ability to directly access the epithelium of the genitourinary (GU) tract via the urethra provides a practical means to implement these various gene therapy approaches. We thus explored vector strategies to accomplish direct in vivo transduction of GU epithelium. Initially, three human (HT 1197, HT 1376, T24) and one mouse (MBT-2) TCC cell lines were transduced using a recombinant adenoviral vector expressing the firefly luciferase reporter gene, rAd-CMV-Luc. In these studies, reporter gene expression was found to be significantly elevated above background for all four cell lines. Of note, the TCC cell lines HT 1197 and HT 1376 showed expression levels comparable with the cervical carcinoma cell line HeLa, a cell line previously shown to be highly susceptible to recombinant adenovirus-mediated gene transduction. An in vitro time course for T24 and MBT-2 using rAd-CMV-Luc showed peak expression 1 day after transduction for the T24 line and 3 days after transduction for the MBT-2 line, with detectable levels of expression persisting for at least 7 days. As a next step, human and mouse primary tissue deriving from the GU epithelium were transduced using rAd-CMV-Luc. In this assay, luciferase expression levels significantly above background were observed in both instances.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenoviruses, Human↗
A carcinoembryonic antigen polynucleotide vaccine has in vivo antitumor activity.
We have constructed a plasmid DNA encoding the full-length cDNA for human carcinoembryonic antigen (CEA) driven by the cytomegalovirus early promoter/enhancer and demonstrated that this plasmid can function as a polynucleotide vaccine. The immune response elicited by the CEA polynucleotide vaccine is dose and schedule dependent. There appears to be a threshold dose of 50 micrograms capable of inducing CEA-specific lymphoblastic transformation, lymphokine release, and antibody response. Doses of 10 micrograms were significantly less effective. When 50-micrograms doses are employed, thrice weekly or weekly vaccination schedules more reliably elicit CEA-specific immune responses by day 43 than does an every-3-weeks schedule. Furthermore the CEA polynucleotide vaccine can immunoprotect against challenge with syngeneic CEA-transduced colon carcinoma cells as early as 3 weeks after the first vaccination. Studies are ongoing to demonstrate the ability of CEA polynucleotide vaccination to treat pre-existing syngeneic mouse colon and breast carcinomas expressing human CEA.
Animals↗
Addition of a short peptide ligand to the adenovirus fiber protein.
A major concern associated with the use of recombinant adenoviral vectors is that viral receptors are found on the surface of many cell types and systemic in vivo delivery of the viral vector could result in uncontrolled and widespread expression of therapeutic molecules in many tissues. To construct a cell-type specific recombinant adenoviral vector, a new binding specificity must be added to the virus, and the endogenous binding specificity of the virus must be ablated. In order to introduce a new binding specificity to recombinant adenoviral vectors, the coding sequence of a physiological ligand, the terminal decapeptide of the gastrin releasing peptide (GRP), was placed at the 3' end of the coding sequence of the adenovirus type 5 fiber gene. The resulting fiber-GRP fusion protein was expressed using a T7 vaccinia expression system and has been shown to assemble protein trimers whose quaternary structure is indistinguishable from that of wild-type protein. The fiber-GRP fusion protein was correctly transported to the nucleus of HeLa cells immediately after synthesis. The added GRP ligand in the fiber-GRP fusion protein was accessible to binding by an anti-GRP antibody in both the monomeric and trimeric forms of the chimeric protein. These studies suggest that new cell type specificities for adenovirus binding might be introduced by genetic fusion of peptide ligands on to the carboxyl terminus of the adenovirus fiber protein.
Adenoviruses, Human↗
Adenoviral-mediated delivery of the herpes simplex virus thymidine kinase gene selectively sensitizes human ovarian carcinoma cells to ganciclovir.
One strategy used for gene therapy of cancer is molecular chemotherapy. This approach is based on selective expression of an encoded toxin in cancer cells to achieve their eradication. One potential advantage of this strategy derives from a phenomenon, termed the bystander effect, whereby only a fraction of cells needs to be transduced to eradicate a tumor population. Despite the theoretical advantages of this phenomenon, it has only been described in a few cellular targets. Therefore, we undertook strategies to develop a molecular chemotherapy approach for ovarian carcinoma utilizing the herpes simplex virus thymidine kinase (HSV-TK) gene. Initially, we established that human ovarian carcinoma cell lines could be transduced at high efficiency with adenoviral vectors encoding reporter genes. We next determined that the human ovarian cell line SKOV3 could exhibit bystander killing by stably transducing it to express HSV-TK and performing cell mixing experiments with varying percentages of HSV-TK-expressing and HSV-TK-nonexpressing cells. Based on these findings, we constructed a recombinant adenovirus encoding HSV-TK and utilized it to induce human ovarian carcinoma cell lines to the sensitizing effects of ganciclovir. In addition, primary cultures of ovarian carcinoma cells were found to be highly transducible with recombinant adenoviral vectors and could be induced to the sensitizing effects of ganciclovir after induction of HSV-TK expression by the adenoviral vector. These studies indicate that molecular chemotherapy using a recombinant adenoviral vector expressing HSV-TK may provide a rational strategy for human ovarian carcinoma.
Adenoviridae↗