PubMed Health⌕ Search

Biomedical subjects

D T Curiel

Publications and source records attributed to D T Curiel.

At least 127 records · Page 7Linked to original sources
PubMed1999

Genetic modification of liver grafts with an adenoviral vector encoding the Bcl-2 gene improves organ preservation.

BACKGROUND: Liver function after transplantation is determined by the quality of the donor organ and the influences of preservation, flush, and reperfusion injury. In this regard, cell death (apoptosis) plays an important role in organ preservation and rejection. Therefore, we examined the possibility of genetic modification of the liver graft with a recombinant adenovirus vector encoding the Bcl-2 gene to reduce apoptosis during the preservation time. METHODS: Liver grafts from C57B1/6 mice were procured and preserved using standard techniques. A replication defective adenovirus vector (deltaE1) containing the human Bcl-2 gene (AdCMVhBcl-2) was developed in our laboratory. An adenovirus vector encoding an irrelevant gene (Escherichia coli beta-galactosidase) was used as a control. Each mouse received 1 x 10(9) plaque forming units administered i.v. 48 hr before the liver procurement. Analyses of liver enzyme activities were determined in the preservation solution. Apoptosis in liver biopsies was determined by DNA fragmentation with an in situ histochemical assay. RESULTS: Immunohistochemical analysis and RT-PCR confirmed the expression of hBcl-2 in the grafts. Grafts from livers expressing hBcl-2 showed significant reduction of the aspartame amino transferase (AST) and lactate dehydrogenase (LDH) release compared with grafts from the control groups. After rewarming, significant cytoprotection was also observed in grafts from animals treated with AdCMVhBcl-2. Histological analysis correlated with the hepatocellular injury determined with transaminases and LDH in the preservation solution. Significant reduction in the number of apoptotic cells was observed in grafts expressing hBcl-2. CONCLUSIONS: We have demonstrated a novel approach to reducing the preservation injury to liver grafts with the human Bcl-2 gene. This approach may allow a longer preservation time, potentially reduce the incidence of primary nonfunction, decrease the immunogenicity of the cold injured organ, and increase the safer use of "marginal" liver grafts.

Adenoviridae↗
PubMed1999

Retargeting to EGFR enhances adenovirus infection efficiency of squamous cell carcinoma.

BACKGROUND: Adenovirus-mediated gene therapy has been used for squamous cell carcinoma of the head and neck (SCCHN), but the in vivo efficacy has been limited by a lack of tissue specificity and low infection efficiency. We are interested in improving cancer gene therapy strategies using targeted adenovirus vectors. OBJECTIVE: To determine if the infection efficiency of adenovirus-mediated gene transfer to SCCHN cells could be enhanced by retargeting to the epidermal growth factor receptor (EGFR), which is known to be overexpressed in these tumors. DESIGN: Epidermal growth factor receptor retargeting in SCCHN cells was accomplished with a bispecific antibody that recognized the knob domain of adenovirus as well as EGFR. Using this retargeting schema, we compared the infection efficiency and specificity of unmodified and EGFR-retargeted adenovirus. RESULTS: Squamous cell carcinoma of the head and neck cell lines were shown to be infected by adenovirus with low efficiency, which is likely because of the low level of adenovirus receptor expressed in the SCCHN cells. Epidermal growth factor receptor retargeting markedly enhanced transduction in both SCCHN cell lines and primary tumor tissue, as indicated by the elevated levels of reporter gene expression. Furthermore, retargeting enhanced infection of tumor tissue compared with normal tissue from the same patient. CONCLUSIONS: Epidermal growth factor receptor retargeting enhanced adenovirus infection of SCCHN cells and, in doing so, augments the potency of the vector. This modification makes the vector potentially more valuable in the clinical setting.

Adenoviridae↗
PubMed1999

An advanced generation of adenoviral vectors selectively enhances gene transfer for ovarian cancer gene therapy approaches.

OBJECTIVE: We hypothesized that incorporation of an integrin binding Arg-Gly-Asp (RGD)-containing peptide to the HI loop of the adenovirus fiber knob would allow enhanced, coxsackie-adenovirus receptor-independent gene transfer by modified Ad vector in the context of ovarian cancer. METHODS: Ovarian cancer cell lines, primary ovarian cancer cells, primary tumor explants, and mesothelial tissue were transfected with luciferase encoding adenovirus (AdCMVLuc) or a genetically modified adenovirus (Ad5lucRGD) which contained an RGD motif within the HI loop of the knob. The luciferase activity was measured and the transduction efficiencies of both viruses were compared. RESULTS: In all established ovarian cell lines and primary tumor cell samples there was dramatically augmented gene transfer observed with the Ad5lucRGD compared to AdCMVLuc. The enhanced gene transfer in ovarian cancer cell lines ranged from 2.5- to 471.6-fold, in ascites samples from 26.1- to 64.0-fold, and in tumor explants from 1.6- to 11.1-fold. Although gene transfer to normal mesothelial tissue was slightly augmented by RGD retargeting, the level of gene transfer was much lower than that seen in ovarian cancer cells. CONCLUSION: This study demonstrates that genetically altered adenoviruses with modified tropism are capable of more efficient gene transfer in the context of ovarian cancer. The higher level of transfer with respect to peritoneal mesothelium can be exploited to enhance the therapeutic index of interventions using adenoviral vectors. Studies are warranted, therefore, to determine the in vivo utility of this targeted vector approach in the context of gene therapeutic strategies for cancer of the ovary.

Adenoviridae↗
PubMed1999

Chimeric viral vectors--the best of both worlds?

Gene therapy to correct defective genes requires efficient gene delivery and long-term gene expression. The vector systems currently available have not allowed the simultaneous provision of both of these goals. Several groups are now developing chimeric viral vector systems that incorporate the favorable attributes of two different viral vectors. These chimeric vectors might allow the goals for specific gene therapy applications to be realized.

Animals↗
PubMed1999

A system for the propagation of adenoviral vectors with genetically modified receptor specificities.

The development of genetically modified adenovirus (Ad) vectors with specificity for a single cell type will require both the introduction of novel tropism determinants and the ablation of endogenous tropism. Consequently, it will not be possible to exploit the native cellular entry pathway in the propagation of these targeted Ad vectors. Based on the concept that Ad enters cells by a two-step process in which a primary receptor serves as a high affinity binding site for the Ad fiber knob, with subsequent internalization mediated by alpha v integrins, we designed two artificial primary receptors. The extracellular domain of one of these synthetic receptors was derived from a single-chain antibody (sFv) with specificity for Ad5 knob, while the second receptor consisted of an icosapeptide identified by biopanning a phage display library against Ad5 knob. Expression of either of these artificial virus-binding receptors in fiber receptor-negative cells possessing alpha v integrins conferred susceptibility to Ad infection. We then created a novel mechanism for cell binding by genetically modifying both the vector and the target cell. In this approach, six histidine (His) residues were incorporated at the C-terminal of the Ad fiber protein. The resultant Ad vector was able to infect nonpermissive cells displaying the cognate artificial receptor, containing an anti-His sFv. This strategy, comprising a genetically engineered Ad virion and a modified cell line, should be useful in the propagation of targeted Ad vectors that lack the ability to bind the native fiber receptor.

Adenoviridae↗
PubMed1999

Tumor-specific gene transfer via an adenoviral vector targeted to the pan-carcinoma antigen EpCAM.

The utility of adenoviral vectors for cancer therapy is limited due to their lack of specificity for tumor cells. In order to target adenovirus to tumor, the natural tropism of the adenovirus should be ablated and replaced by a tumor-specific binding domain. To this end, a neutralizing anti-fiber antibody conjugated to an anti-EpCAM antibody was created that targets the adenovirus to the EpCAM antigen present on tumor cells. The EpCAM antigen was chosen as the target because this antigen is highly expressed on a variety of adenocarcinomas of different origin such as breast, ovary, colon and lung, whereas EpCAM expression is limited in normal tissues. In these studies, the EpCAM-targeted adenovirus was shown to infect specifically cancer cell lines of different origin expressing EpCAM such as ovary, colon and head and neck. Gene transfer was blocked by excess anti-EpCAM antibody and dramatically reduced in EpCAM negative cell lines, thus showing the specificity of the EpCAM-targeted adenovirus. Importantly, infection with targeted adenovirus was independent of CAR, which is the natural receptor for adenovirus binding, since blocking of CAR with recombinant fiber knob did not affect infection with targeted adenovirus. Apart from the cancer cell lines, the efficacy of targeted viral infection was studied in freshly isolated primary human colon cancer cells. As colon cancer predominantly metastasizes to liver, and adenovirus has a high tropism for hepatocytes, we also sought to determine if the EpCAM-targeted adenovirus showed reduced infectivity of human liver cells. The bispecific antibody could successfully mediate gene transfer to primary human colon cancer cells, whereas it almost completely abolished infection of liver cells. This work thus demonstrates that EpCAM-targeted adenoviral vectors can be specifically directed to a wide variety of adenocarcinomas. This approach may prove to be useful for selective gene therapy of cancer.

Adenocarcinoma↗
PubMed1999

Therapeutic efficacy of an adenovirus-mediated anti-H-ras ribozyme in experimental bladder cancer.

Ras oncogenes are thought to play a critical role in cellular proliferation and tumorigenesis. Reversal of the malignant phenotype, inhibition of tumor growth, and decreased tumorgenicity have been demonstrated with the use of anti-H-ras ribozymes. In this study, the therapeutic efficacy of a hammerhead ribozyme targeting the mutated H-ras oncogene was investigated in an experimental bladder cancer model using a recombinant adenovirus as delivery vehicle. Tumors were established in nude mice by subcutaneous injection of EJ human bladder carcinoma cells harboring a point mutation of the H-ras gene. The tumors were treated with intralesional injections of an adenovirus expressing an anti-H-ras ribozyme (rAd-Hras Rz) by different schedules at serial titers, and the tumor inhibition efficacy was analyzed. The viral infection efficacy and kinetics of ribozyme expression were also evaluated. Intralesional injection of rAd-Hras Rz resulted in significant antineoplastic effects in a dose-dependent fashion. Complete regression of the tumor was achieved by rAd-Hras Rz in several cases without recurrence during the 50-day observation period. Although there was moderate vector-associated cytotoxicity in this cell line, complete regressions were not observed in the cases treated with control adenovirus vectors or vectors expressing an inactive anti-H-ras ribozyme or anti-H-ras antisense oligonucleotides. These results suggest the efficacy of a ribozyme-encoding adenovirus in the experimental gene therapy of human bladder cancer.

Adenoviridae↗
PubMed1999

Reduction of ischemia-reperfusion injury of the liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic Bcl-2 gene.

OBJECTIVE: To examine the possibility of reducing ischemia-reperfusion injury (I/R injury) to the mouse liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic human Bcl-2 gene. SUMMARY BACKGROUND DATA: Ischemia-reperfusion injury has been demonstrated in a number of clinically relevant diseases such as myocardial infarction, cerebrovascular disease, sepsis, peripheral vascular disease, and organ transplantation. In this regard, apoptosis plays a central role. METHODS: Normal C57BL/6 mice were used. An adenovirus (deltaE1) vector containing the human Bcl-2 gene was developed in the authors' laboratory. An adenovirus vector encoding an irrelevant gene (beta-galactosidase, AdCMVLacZ) was used as a control. Taking advantage of the hepatotropic properties of adenovirus vectors, gene transfer was performed with 1 x 10(9) plaque-forming units by intravenous tail injection, 48 hours before the ischemic injury. Ischemic-reperfusion injury was induced by temporal and segmental occlusion of hepatic blood flow. Aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase activity was measured using standard assays. Liver biopsies were obtained before and 6 hours after I/R injury for morphologic assessment, and apoptosis was determined in situ with a histochemical assay. RESULTS: The expression of AdCMVhBcl-2 vector was confirmed by reverse transcription-polymerase chain reaction and functionally validated in apoptotic studies in endothelial cells. Expression of the Bcl-2 gene protects against I/R injury, as shown by a significant decrease in transaminases (p < 0.05) and necrosis and apoptosis (p < 0.001), and permanent survival (p < 0.0001), compared with sham-operated animals and animals treated with AdCMVLacZ. CONCLUSIONS: Genetic modification of the liver to induce cytoprotection has potential applications to prevent I/R injury to the liver in surgical interventions, including liver transplantation.

Adenoviridae↗
PubMed1999

Strategies to adapt adenoviral vectors for targeted delivery.

The utility of current generation adenoviral vectors for targeted, cell-specific gene delivery is limited by the promiscuous tropism of the parent virus. To address this issue, we have developed both genetic and immunologic methods to alter viral tropism. Immunologic retargeting has been achieved via conjugates comprised of an antifiber knob Fab and a targeting moiety consisting of a ligand or antireceptor antibody. Gene delivery by this approach has been accomplished via a variety of cellular pathways including receptors for folate, FGF, and EGF. In addition to cell-specific gene delivery, this strategy has allowed enhanced gene delivery to target cells lacking the native adenoviral receptor, CAR. Of note, this specific and extended gene delivery allowed enhanced survival in murine models of human carcinoma via cancer gene therapy. Genetic strategies to alter adenoviral tropism have included both fiber modification and fiber replacement. In the former, we have identified the HI loop of fiber as a propitious locale for introduction of heterologous peptides. Incorporation of an RGDC peptide at this locale allowed gene delivery via cellular integrins with dramatic efficiency augmentations. As a strategy to achieve both new tropism as well as to ablate native tropism, methods have been developed to replace the fiber protein with heterologous motif which preserves the key trimeric quaternary structure of fiber and allows for propagation. Such a fiber-replacement virus has been rescued and has demonstrated capacities consistent with its utility as a novel vector agent. These strategies have allowed the achievement of cell-specific gene delivery via adenoviral vectors and thus have the potential to enhance the utility of this vector agent.

Adenoviridae↗
PubMed1999

Adenovirus-mediated gene expression in vivo is enhanced by the antiapoptotic bcl-2 gene.

An adenovirus vector encoding the human Bcl-2 gene (hBcl-2) was derived. In vivo expression of hBcl-2 in murine livers enhanced and prolonged adenovirus-mediated gene expression. Furthermore, in the hBcl-2-treated group a significant reduction in the apoptosis induced by the adenovirus vector was observed. Thus, the cytoprotection of the vector-infected cells with antiapoptotic genes appears promising for successful in vivo gene therapy.

Adenoviruses, Human↗
PubMed1999

Bcl-2 and Bcl-XL serve an anti-inflammatory function in endothelial cells through inhibition of NF-kappaB.

To maintain the integrity of the vascular barrier, endothelial cells (EC) are resistant to cell death. The molecular basis of this resistance may be explained by the function of antiapoptotic genes such as bcl family members. Overexpression of Bcl-2 or Bcl-XL protects EC from tumor necrosis factor (TNF)-mediated apoptosis. In addition, Bcl-2 or Bcl-XL inhibits activation of NF-kappaB and thus upregulation of proinflammatory genes. Bcl-2-mediated inhibition of NF-kappaB in EC occurs upstream of IkappaBalpha degradation without affecting p65-mediated transactivation. Overexpression of bcl genes in EC does not affect other transcription factors. Using deletion mutants of Bcl-2, the NF-kappaB inhibitory function of Bcl-2 was mapped to bcl homology domains BH2 and BH4, whereas all BH domains were required for the antiapoptotic function. These data suggest that Bcl-2 and Bcl-XL belong to a cytoprotective response that counteracts proapoptotic and proinflammatory insults and restores the physiological anti-inflammatory phenotype to the EC. By inhibiting NF-kappaB without sensitizing the cells (as with IkappaBalpha) to TNF-mediated apoptosis, Bcl-2 and Bcl-XL are prime candidates for genetic engineering of EC in pathological conditions where EC loss and unfettered activation are undesirable.

Animals↗
PubMed1999

In vivo localization of [(111)In]-DTPA-D-Phe1-octreotide to human ovarian tumor xenografts induced to express the somatostatin receptor subtype 2 using an adenoviral vector.

Adenoviral vectors, encoding genes for cell surface antigens or receptors, have been used to induce their high level expression on tumor cells in vitro and in vivo. These induced antigens and receptors can then be targeted with radiolabeled antibodies or peptides for potential radiotherapeutic applications. The purpose of this study was to determine a dosing schema of an adenoviral vector encoding the human somatostatin receptor subtype 2 (AdCMVhSSTr2) for achieving the highest tumor localization of [(111)In]-DTPA-D-Phe1-octreotide, which binds to this receptor, in a human ovarian cancer model as a prelude to future therapy studies. AdCMVhSSTr2 was produced and used to induce hSSTr2 on A427 human nonsmall cell lung cancer cells and on SKOV3.ipl human ovarian cancer cells in vitro, as demonstrated by competitive binding assays using [125I]-Tyr1-somatostatin and [(111)In]-DTPA-D-Phe1-octreotide. Mice bearing i.p. SKOV3.ip1 tumors administered 1 x 10(9) plaque-forming units of AdCMVhSSTr2 i.p. 5 days after tumor cell inoculation, followed by an i.p. injection of [(111)In]-DTPA-D-Phe1-octreotide 2 days later, showed a range of 15.3-60.4% median injected dose/gram (ID/g) in tumor at 4 h after injection compared with 3.5% ID/g when [125I]-Tyr1-somatostatin was administered and 0.3% ID/g when the negative control peptide [125I]-mIP-bombesin was administered. Mice administered a control adenoviral vector encoding the gastrin-releasing peptide receptor did not have tumor localization of [(111)In]-DTPA-D-Phe1-octreotide (<1.6% ID/g), demonstrating specificity of [(111)In]-DTPA-D-Phe1-octreotide for the AdCMVhSSTr2 induced tumor cells. In another set of experiments, the tumor localization of [(111)In]-DTPA-D-Phe1-octreotide was not different 1, 2, or 4 days after AdCMVhSSTr2 injection (31.8, 37.7, and 40.7% ID/g, respectively; P = 0.88), indicating that multiple injections of radiolabeled peptide can be administered with equivalent uptake over a 4-day period. [(111)In]-DTPA-D-Phe1-octreotide tumor localization in animals administered AdCMVhSSTr2 on consecutive days or 2 days apart was 22.4% ID/g and 53.2% ID/g, respectively (P = 0.009) when [(111)In]-DTPA-D-Phe1-octreotide was given 1 day after the second AdCMVhSSTr2 injection. There was no difference in [(111)In]-DTPA-D-Phe1-octreotide localization after a single AdCMVhSSTr2 injection (40.7% ID/g) or two injections of AdCMVhSSTr2 given 1 (45.9% ID/g) or 2 (53.2% ID/g) days apart, where [(111)In]-DTPA-D-Phe1-octreotide was given in each case 4 days after the first AdCMVhSSTr2 injection (P = 0.65). Therefore, two AdCMVhSSTr2 injections did not increase [(111)In]-DTPA-D-Phe1-octreotide tumor localization compared with one injection, which eliminates concerns about an immune response to a second dose of AdCMVhSSTr2. This will be the basis for a therapeutic protocol with multiple administrations of an octreotide analogue labeled with a therapeutic radioisotope.

Adenoviridae↗
PubMed1999

Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector.

In vivo cancer gene therapy approaches for squamous cell carcinoma of the head and neck (SCCHN) based on adenoviral vector-mediated gene delivery have been limited by the suboptimal efficacy of gene transfer to tumor cells. We hypothesized that this issue was due to deficiency of the primary adenoviral receptor, the coxsackie-adenovirus receptor (CAR), on the tumor targets. Studies of CAR levels on SCCHN cell lines confirmed that their relative refractoriness to the adenoviral vector was based on this deficiency. To circumvent this deficiency, we applied an adenoviral vector targeted to a tumor cell marker characteristic of SCCHN. In this regard, integrins of the alpha2beta1 and alpha3beta1 class are frequently overexpressed in SCCHN. Furthermore, these integrins recognize the RGD peptide motif. On this basis, we applied an adenoviral vector genetically modified to contain such a peptide within the HI loop of the fiber protein as a means to alter viral tropism. Studies confirmed that the CAR-independent gene delivery achieved via this strategy allowed enhanced gene transfer efficiencies to SCCHN tumor cells. Importantly, this strategy could achieve preferential augmentation of gene transfer in tumor cells compared with normal cells. The ability to achieve enhanced and specific gene transfer to tumor cells via adenoviral vectors has important implications for gene therapy strategies for SCCHN and for other neoplasms in general.

Adenoviridae↗
PubMed1999

Targeting strategies for cancer radiotherapy.

Novel strategies to increase the therapeutic ratio in clinical radioimmunotherapy studies are needed. Limitations to radioimmunotherapy include bone marrow suppression due to the long circulating half-life of radiolabeled monoclonal antibodies (mAbs) and heterogeneous tumor penetration of the high-molecular-weight mAb. An approach to overcome these problems is the use of genetically engineered mAbs. The engineered mAb discussed in this paper contains a deletion in the constant region of the mAb that increases its tumor penetration and blood clearance compared with the intact mAb. Radiolabeling of this mAb should lead to a similar radiation-absorbed dose to tumor compared with the intact mAb, but reduce the radiation absorbed dose to bone marrow. In addition, low or variable expression of tumor-associated target antigens or receptors may lead to low or heterogeneous tumor uptake of radiolabeled mAbs. This report also discusses a novel approach toward systemic radiotherapy that combines gene transfer techniques (to increase tumor receptor expression) with radiolabeled peptides that target the induced receptor. The radiolabeled peptides achieve good tumor uptake, rapid tumor penetration, and rapid blood clearance. A humanized construct of the CC49 (HuCC49) high-affinity anti-TAG-72 mAb, as well as a construct with the CH2 region deleted (HuCC49deltaCH2), were labeled with 131I and 177Lu. Biodistribution of the radiolabeled constructs was evaluated 24 h after regional i.p. injection in athymic nude mice bearing i.p. LS174T human colon cancer xenografts. The 131I-HuCC49deltaCH2 showed a median tumor uptake of 5.5% ID/g which was similar to that of 131I-HuCC49 at 5.2% ID/g. However, the median blood concentration of 131I-HuCC49deltaCH2 was 0.2% ID/g which was significantly lower than 0.8% ID/g for 1311-HuCC49. The uptake of the constructs in other normal tissues were similar. The 177Lu-HuCC49deltaCH2 showed a median tumor uptake of 9.4% ID/g, which was slightly higher than that of 177Lu-HuCC49 at 7.9% ID/g. The median blood concentration of 177Lu-HuCC49deltaCH2 was 0.2% ID/g, which was significantly lower than 0.4% ID/g for 177Lu-HuCC49. The uptake of the antibody constructs in other normal tissues were similar except for the kidney. The tumor:blood ratios of 177Lu-HuCC49 and 177Lu-HuCC49deltaCH2 were 19.4 and 60.2, respectively, at 24 h after injection. The purpose of the second aspect of the study was to determine the biodistribution of 64Cu-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA)-octreotide in a human ovarian cancer model induced to express human somatostatin receptor subtype 2 (SSTr2) using gene transfer techniques as a prelude to future therapy studies. Mice bearing i.p. SKOV3.ip1 tumors transduced with an adenoviral vector encoding the cDNA for SSTr2 (AdSSTr2) and injected i.p. with 64Cu-TETA-octreotide showed a median uptake of 24.3% ID/g in tumor at 4 h postinjection compared with 4.9% ID/g at 18 h after injection. Also, tumor uptake of 64Cu-TETA-octreotide at 4 h was not significantly different when administered either 2 or 4 days after injection of AdSSTr2 (P = 0.076). 64Cu-TETA-octreotide should be useful for targeted radiotherapy against tumors that are genetically induced to express high levels of SSTr. These two novel targeting strategies show promise for improved cancer radioimmunotherapy.

Animals↗