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R G Crystal

Publications and source records attributed to R G Crystal.

At least 91 records · Page 5Linked to original sources
PubMed1998

Reversal of CPT-11 resistance of lung cancer cells by adenovirus-mediated gene transfer of the human carboxylesterase cDNA.

To evaluate the concept that transfer of the human carboxylesterase (CE) gene will overcome the drug resistance of a solid tumor to CPT-11 (irinotecan), we used an adenovirus vector (AdCMV.CE) carrying human CE cDNA to infect CPT-11-resistant A549 human adenocarcinoma cells (A549/CPT) in vitro and in vivo and evaluated cell growth over time. The A549/CPT cells, selected by stepwise and continuous exposure of parental A549 cells to CPT-11 over 10 months, had a 6-fold resistance to CPT-11 and 42% CE activity in comparison with parental A549 cells. AdCMV.CE infection resulted in an increase in functional CE protein in resistant cells in vitro that was sufficient to convert CPT-11 to its active metabolite, SN-38, and effectively suppressed resistant cell growth in vitro in the presence of CPT-11. When AdCMV.CE was directly injected into established s.c. resistant A549-based tumors in nude mice receiving CPT-11, there was a 1.8-fold reduction in tumor size at day 20 compared to that of controls (P < 0.05). These observations suggest that adenovirus-mediated gene transfer of the human CE gene and concomitant administration of CPT-11 may have potential as a strategy for local control of acquired CPT-11 resistance of solid tumors.

Adenocarcinoma↗
PubMed1998

Intermittent, repetitive corticosteroid-induced upregulation of platelet levels after adenovirus-mediated transfer to the liver of a chimeric glucocorticoid-responsive promoter controlling the thrombopoietin cDNA.

For many in vivo gene therapy clinical applications, it is desirable to control the expression of the transferred transgene using pharmacologic agents. To evaluate the feasibility of accomplishing this using corticosteroids, pharmacologic agents widely used in clinical medicine, we constructed replication deficient adenoviral (Ad) vectors containing an expression cassette with a chimeric promoter comprised of five glucocorticoid response elements (GRE) and the chloramphenicol acetyltransferase reporter gene (AdGRE.CAT) or the murine thrombopoietin cDNA (AdGRE.mTPO). In vitro studies showed the vectors functioned as expected, with marked glucocorticoid-induced upregulation of the CAT or mTPO transgenes. To evaluate the inducibility of the GRE promoter in vivo, the AdGRE. CAT vector was administered intravenously to C57B1/6 mice, and CAT activity was quantified in liver before and after intraperitoneal administration of dexamethasone. The GRE promoter activity was dependent on the dexamethasone dose, with a 100-fold increase in CAT expression with 50 microg dexamethasone, similar to the levels observed in vivo with the Rous sarcoma virus long terminal repeat constitutive promoter. After dexamethasone administration, maximum CAT activity was observed at day 2, with a slow decline to baseline levels by 2 weeks. Based on these observations, we hypothesized that a single administration of an Ad vector-mediated transfer of the chimeric GRE inducible promoter driving the mTPO cDNA would enable repetitive administration of corticosteroids to repetitively upregulate platelet levels for 1 to 2 weeks. The data show that this occurs, with dexamethasone administration every 3 weeks associated with 1-week elevations (at each 3-week interval) of serum mTPO levels, megakaryocyte numbers in bone marrow, and platelet levels fourfold to sixfold over baseline. Thus, with the appropriate promoter, it is possible to use a commonly used pharmacologic agent to upregulate the expression of a newly transferred gene on demand.

Adenoviruses, Human↗
PubMed1998

Gene therapy for oxidant injury-related diseases: adenovirus-mediated transfer of superoxide dismutase and catalase cDNAs protects against hyperoxia but not against ischemia-reperfusion lung injury.

Hyperoxia and ischemia-reperfusion cause profound lung cellular damage mediated, in part, by generation of oxygen radicals. We hypothesized that gene therapy can be used to overcome oxidant injury by augmenting intracellular antioxidant enzymes. Adult rats were injected intratracheally with an adenovirus (Ad) vector encoding human superoxide dismutase (CuZn-SOD) or catalase cDNA, a mixture of both Ad vectors, or a control Ad vector containing no exogenous gene. Expression of human catalase and CuZn-SOD was demonstrated 3 days later in distal lung epithelial cells and alveolar macrophages, using ELISA and immunochemistry. After exposure to 100% O2 for 62 hr, survival was greater in rats injected with the catalase and/or SOD Ad vectors than in control rats. Ischemia-reperfusion injury was evaluated in the isolated perfused lung model. Overexpression of SOD worsened ischemia-reperfusion injury. Interestingly, concomitant overexpression of catalase prevented this adverse effect, but did not protect against ischemia-reperfusion injury. We conclude that Ad-mediated transfer to lungs of both catalase and SOD cDNAs protects from pulmonary O2 toxicity. Absence of protection against ischemia-reperfusion using intratracheal Ad injections may be related to the lack of endothelial protection, despite epithelial expression of catalase and SOD.

Adenoviridae↗
PubMed1998

Similarity of strain- and route-dependent murine responses to an adenovirus vector using the homologous thrombopoietin cDNA as the reporter genes.

Replication-deficient adenovirus (Ad) vectors are effective in transferring genes in vivo, but their use is associated with significant variation in the extent and/or duration of expression observed among different strains of experimental animals and different routes of administration of the vector. We have minimized the variables of the heterologous transgene and animal-to-animal variation by constructing an Ad vector encoding murine thrombopoietin (mTPO, AdmTPO), a homologous protein that induces a physiologic response (elevation of blood platelet levels) that can be followed sequentially over time in the same animal. Using the C57BL/6 and BALB/c stains, liver administration was accomplished by intravenous administration and skeletal muscle administration by direct injection. Despite the use of a homologous cDNA as a transgene, the Ad genome was rapidly lost from the liver after intravenous administration over the first 1 to 2 weeks, with no difference in pattern of decline between the C57BL/6 and BALB/c strains. Both strains exhibited a cytotoxic T lymphocyte (CTL) response directed against the AdmTPO vector. Consistent with the decline in vector genome over time, the initial high levels of mTPO mRNA in the liver declined to an undetectable level within 2 weeks. Platelet counts peaked at 8- to 10-fold above baseline within the first 2 weeks, and then gradually declined, returning to normal level by 50 to 60 days. Intravenous administration of the AdmTPO vector to beta2-microglobulin-deficient mice resulted in a longer persistence of elevated platelets levels, although the eventual return of platelet levels to normal in these mice suggests the elimination of the Ad vector cannot be explained solely by CTL response. Although the intramuscular administration of the AdmTPO vector resulted in platelet levels with a lower peak and minor differences over time compared with the intravenous route, the C57BL/6 and BALB/c strains demonstrated the same rapid loss of Ad genome and mTPO mRNA levels in the muscle as in the liver. Together, these observations suggest that simplifying the experimental design by eliminating the variable of host response to a heterologous transgene, and by following the consequences of gene transfer in the same animals over time, there can be remarkable similarity in strain- and route-dependent responses to an Ad vector.

Adenoviridae↗
PubMed1998

In vivo human carboxylesterase cDNA gene transfer to activate the prodrug CPT-11 for local treatment of solid tumors.

To evaluate the concept that in vivo transfer of the human carboxylesterase gene will confer sensitivity of a solid tumor to the prodrug CPT-11 (irinotecan), we constructed an adenovirus vector (AdCMV.CE) carrying the human carboxylesterase gene driven by the cytomegalovirus (CMV) promoter, infected A549 human lung adenocarcinoma cells in vitro and in vivo, and evaluated cell growth over time. AdCMV.CE produced a functional carboxylesterase protein in A549 cells in vitro and in vivo as evidenced by ability of lysates from the infected cells to convert CPT-11 to its active metabolite SN-38. The AdCMV.CE vector effectively suppressed A549 cell growth in vitro in the presence of CPT-11. Cell mixing studies demonstrated that when as few as 10% of cells expressed the human carboxylesterase gene, there was bystander growth suppression in the presence of CPT-11. Consistent with these in vitro observations, when AdCMV.CE was directly injected into established subcutaneous A549 tumors in nude mice receiving CPT-11, there was 35% reduction in tumor size at day 27 compared to controls, and a 41% reduction at day 34 (P < 0.01, both comparisons to controls). Similar observations were made with the cell line H157 and HeLa. These observations suggest that local gene transfer of the human carboxylesterase gene and concomitant local administration of CPT-11 may have potential as a strategy for control of the growth of solid tumors.

Adenoviridae↗
PubMed1998

High-efficiency gene transfer into ex vivo expanded human hematopoietic progenitors and precursor cells by adenovirus vectors.

Replication-deficient adenoviral vectors (AdVec), which infect cycling and noncycling cells with high efficiency, low toxicity, and ease of delivery, provide ideal vehicles to study the expression of regulatory genes controlling different stages of hematopoiesis. To examine the infection efficiency of AdVec in hematopoietic precursor and progenitor cells, we used a replication-deficient adenovector expressing the humanized form of the cDNA for green fluorescent protein (AdGFP), permitting assessment of infection efficiency and kinetics of transgene expression in viable hematopoietic cells using flow cytometry and fluorescence microscopy. Flow-cytometric analysis of ex vivo expanded hematopoietic precursor cells infected with a multiplicity of infection (MOI) of 100 of AdGFP show that 78% of megakaryocytic (CD41a+ and CD42b+) cells, 82% of dendritic (CD1a+) cells, 41% of RBC precursors (glycophorin A+), and 32% of monocytic (CD14(+)) cells expressed GFP. Nineteen percent +/- 1% of freshly isolated CD34(+) cells from peripheral blood leukapheresis products infected under the same conditions expressed GFP. Morphologic evaluation of ex vivo expanded, AdGFP-infected CD34(+) cells showed normal maturation. The functional capacity of AdGFP-infected CD34(+) cells was analyzed by quantifying clonogeneic efficiency and proliferative capacity. Infection of CD34(+) progenitor cells with MOIs of 1 to 100 did not impair clonogeneic efficiency of CD34(+ )cells. However, MOI greater than 100 resulted in a significant inhibition of colony-forming unit-granulocyte/granulocyte-macrophage (CFU-G/GM) formation. In sequential dilution expansion over 3 weeks (Delta assay), the cytokine-driven proliferative potential of CD34(+) cells was not impaired following exposure to AdGFP at MOIs of 1 to 1,000. The GFP+ population expanded 10- to 15-fold at high MOIs (500 to 1,000), indicating multiple copies of the transgene in the initially infected CD34(+) cells, which were expressed in subsequent progenies. These data show that AdVec deliver transgenes with high efficiency and low toxicity to hematopoietic progenitor and precursor cells. Introduction of marker genes such as GFP into hematopoietic cells by AdVec will provide a valuable system for study of development, homing, and trafficking of hematopoietic precursor and progenitor cells in vitro and in vivo. Furthermore, these results provide insights into the design of gene therapy strategies for treatment of hematologic disorders by AdVec.

Adenoviridae↗
PubMed1998

Regional suppression of tumor growth by in vivo transfer of a cDNA encoding a secreted form of the extracellular domain of the flt-1 vascular endothelial growth factor receptor.

Vascular endothelial growth factor (VEGF), a potent angiogenic mediator, is overexpressed in most solid tumors. On the basis of the knowledge that solid tumor growth beyond a small volume is critically dependent on angiogenesis, and that adenovirus (Ad) vectors can mediate efficient in vivo gene transfer and expression, we hypothesized that Ad-mediated transfer of a secreted form of the extracellular domain of the flt-1 VEGF receptor (Adsflt) would suppress tumor growth on a regional basis. To evaluate this concept, three tumor models were examined using a murine colon carcinoma cell line and syngeneic BALB/c mice. First, mice with preestablished splenic CT26.CL25 tumors and liver metastases were given Adsflt on AdNull intravenously and, after 15 days, spleens and livers were harvested to quantify tumor burden. Adslft-treated animals had minimal residual splenic tumors and liver metastases; in contrast, control animals had bulky splenic tumors and extensive liver metastases (p < 0.003). Second, mice with preestablished lung metastases showed a significant reduction in pulmonary metastases with regionally administered Adslft (intratracheal, p < 0.02) but not when the vector was systemically administered (intravenous, p > 0.9). Finally, mice with primary subcutaneous tumors treated with intratumoral administration of Adslft showed significant tumor suppression (p < 0.05) not observed in AdNull-treated mice or mice given Adslft intravenously (p > 0.3). We conclude that Ad-mediated in vivo regional delivery of a secreted form of the extracellular domain of the flt-1 VEGF receptor can effectively inhibit regional tumor growth, a strategy that may provide a means to control tumor growth within the treated organ without the risk of systemic antiangiogenesis.

Adenoviridae↗
PubMed1998

Gene therapy strategies for tumor antiangiogenesis.

Based on the concept that solid tumors cannot grow without the generation of new blood vessels, there is growing interest in the use of antiangiogenesis agents to inhibit tumor growth. This review summarizes the concepts of using gene transfer vectors to provide high concentration of antiangiogenic proteins within an organ. While there are many challenges that must be met before antiangiogenesis can be used to effectively treat human tumors, gene transfer strategies have the potential to provide sustained, high, local concentrations of antiangiogenic mediators specifically targeted to organs containing tumors, minimizing systemic toxicity. Antiangiogenesis gene therapy strategies will most likely be effective in a state of low tumor burden, where this "genetic tourniquet" can provide trans (i.e., acting in the extracellular milieu as opposed to within tumor cells) suppression of the growth of endothelial cells in the milieu of micrometastases.

Animals↗
PubMed1998

Fluorescent virions: dynamic tracking of the pathway of adenoviral gene transfer vectors in living cells.

The pathogenic agent, adenovirus (Ad), has taken on a new role as a vector for gene transfer in both laboratory and clinical settings. To help understand the intracellular pathways and fate of Ad gene transfer vectors, we covalently conjugated fluorophores to E1-, E3- Ad vectors and used quantitative fluorescence microscopy to assess essential steps of Ad vector gene transfer to the A549 human epithelial lung cell line including binding, internalization, escape from endosomes, translocation to the nucleus, dissociation of capsids and gene expression. The data demonstrate that Ad internalizes with a t1/2 2.5 min, breaks out of endosomes early, likely prior to endosome-endosome fusion, exhibits sustained, intracellular velocities averaging 0.58 microm/sec, and translocates to the nucleus with >80% of internalized fluorophore demonstrating nuclear localization within 60 min of infection. Interestingly, 24 hr after infection, half of the initially internalized fluorescence was detected but lacked nuclear localization, suggesting that the capsid is released from the nucleus and is likely degraded. Fluorescent labeling of virions provides a novel quantitative, morphological strategy to characterize the interaction of gene transfer vectors with the intracellular environment.

Adenoviridae↗
PubMed1998

Adenovector-mediated expression of human thrombopoietin cDNA in immune-compromised mice: insights into the pathophysiology of osteomyelofibrosis.

Thrombopoietin (TPO) cDNA can be effectively delivered in vivo by adenovectors. Immune normal mice (BALB/c) and syngeneic mice with variable degrees of immune dysfunction nu, SCID, and NOD-SCID) were treated with an adenovirus vector expressing the human TPO cDNA (AdTPO). Platelet peaks were significantly higher in SCID and NOD-SCID mice compared with BALB/c and nu mice. Human plasma TPO concentration correlated with the platelet counts. SCID and NOD-SCID mice exhibited also granulocytosis and increased numbers of hemopoietic progenitors in bone marrow. Following platelet peak, BALB/c mice developed autoantibodies against murine TPO leading to thrombocytopenia and depletion of megakaryocytes and hemopoietic progenitors in bone marrow. AdTPO-treated SCID mice developed osteomyelofibrosis and extramedullary/extrasplenal hemopoiesis. In contrast, NOD-SCID mice with a similar magnitude of TPO overexpression did not show fibrotic changes in bone marrow. We conclude, first, that a chronic high level of TPO overexpression stimulates megakaryocytopoiesis and myelopoiesis leading to thrombocytosis and granulocytosis. Second, increased megakaryocytopoiesis is not sufficient for development of secondary osteomyelofibrosis. The functionally deficient monocytes and macrophages of NOD-SCID mice probably prevented fibrotic marrow changes. Third, immune deficiency enhances expression of adenovirally mediated transgenes, and fourth, xenogeneic transgene delivered by adenovector to a host with normal immune functions may induce loss of immune tolerance and autoimmune phenomenon.

Adenoviruses, Human↗
PubMed1998

Pharmacological expression in rat hepatocytes of a gene transferred by an adenovirus vector enabled by a chimeric promoter containing multiple cyclic adenosine monophosphate response elements.

Using the adenovirus vector AdCF126(CRE8).Luc to deliver an expression cassette containing multiple cyclic adenosine monophosphate (cAMP) response elements driving the luciferase reporter gene, this study is directed toward evaluating the possibility of controlling genes transferred to the liver using pharmacological agents that raise hepatocyte cAMP levels. Infection of primary rat hepatocytes with AdCF126(CRE8).Luc yielded a low level of luciferase activity that was enhanced 16-fold by the addition of forskolin. Direct intrahepatic administration of the Ad vector in C57B1/6 mice resulted in low-level luciferase activity that was increased 76-fold by the administration of theophylline and 8-bromo-cAMP to increase cAMP levels. In contrast, animals receiving intrahepatic administration of a control vector containing a constitutively active Rous sarcoma virus (RSV) viral promoter driving the luciferase gene had no response to elevated cAMP Strikingly, delivery of the vector to the liver by the intravenous route permitted a 258-fold enhancement of liver luciferase activity following administration of the same cAMP-elevating agents. In comparison, a control Ad vector with the RSV promoter was not activated by the elevation in cAMP The maximum luciferase levels achieved by the combination of AdCF126(CRE8).Luc and pharmacological cAMP elevation was 45-fold greater than that with the RSV promoter. These results show the feasibility of using a chimeric promoter to permit pharmacological induction of high-level expression from an expression cassette transferred to the liver with an adenovirus vector, an approach that may be useful in a variety of liver-related gene-transfer strategies.

Adenoviridae↗
PubMed1998

Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart.

OBJECTIVES: Vascular endothelial growth factor (VEGF), a potent angiogenic mediator, can be delivered to targeted tissues by means of a replication-deficient adenovirus (Ad) vector. We hypothesized that direct administration of Ad vector expressing the VEGF121 complementary deoxyribonucleic acid (AdGVVEGF121.10) into regions of ischemic myocardium would enhance collateral vessel formation and improve regional perfusion and function. METHODS: Yorkshire swine underwent thoracotomy and placement of an Ameroid constrictor (Research Instruments & MFG, Corvallis, Ore.) on the circumflex coronary artery. Three weeks later, myocardial perfusion and function were assessed by single photon emission computed tomography imaging (SPECT) with 99mTc-labeled sestamibi and by echocardiography during rest and stress. AdGVVEGF121.10 (n = 7) or the control vector, AdNull (n = 8), was administered directly into the myocardium at 10 sites in the circumflex distribution (10(8) pfu/site). Four weeks later, these studies were repeated and ex vivo angiography was performed. RESULTS: SPECT imaging 4 weeks after vector administration demonstrated significant reduction in the ischemic area at stress in AdGVVEFG121.10-treated animals compared with AdNull control animals (p = 0.005). Stress echocardiography at the same time demonstrated improved segmental wall thickening in AdGVVEGF121.10 animals compared with AdNull control animals (p = 0.03), with AdGVVEGF121.10 animals showing nearly normalized function in the circumflex distribution. Collateral vessel development assessed by angiography was also significantly greater in AdGVVEGF121.10 animals than in AdNull control animals (p = 0.04), with almost complete reconstitution of circumflex filling in AdGVVEGF121.10 animals. CONCLUSIONS: An Ad vector expressing the VEGF121 cDNA induces collateral vessel development in ischemic myocardium and results in significant improvement in both myocardial perfusion and function. Such a strategy may be useful in patients with ischemic heart disease in whom complete revascularization is not possible.

Adenoviridae↗
PubMed1998

Salvage angiogenesis induced by adenovirus-mediated gene transfer of vascular endothelial growth factor protects against ischemic vascular occlusion.

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, and transgene expression from adenovirus vectors can provide in vivo delivery of proteins. On the basis of this knowledge, we hypothesized that local administration of a replication-deficient adenovirus vector expressing complementary DNA for VEGF (AdVEGF) would induce collateral vessel formation in the setting of ischemia that could protect against subsequent acute vascular occlusion. METHODS: Hindlimb ischemia was induced in Sprague-Dawley rats by means of unilateral ligation of the common iliac artery immediately followed by administration of 4 x 10(9)-plaque-forming units VEGF, the control vector AdNull, or phosphate-buffered saline solution into the iliofemoral adipose tissue and thigh muscles. Untreated rats with common iliac ligation were used as an additional control group. RESULTS: Local VEGF expression was observed for 5 days in AdVEGF-treated rats but not in controls. Three weeks after ligation and vector administration, the ipsilateral femoral artery was ligated for a model of an acute vascular occlusion in the setting of preexisting ischemia. Blood flow to the ischemic hindlimb relative to the contralateral hindlimb evaluated with color microspheres demonstrated significantly increased blood flow in the AdVEGF-treated rats compared with each control group (p < 0.0001). Relative blood flow assessed by means of 99mTc-sestamibi radionuclide scans also demonstrated increased blood flow to the ligated hindlimb of AdVEGF-treated rats compared with each control group (p < 0.002). AdVEGF-treated rats also demonstrated increased vascularity in the ligated limb compared with each control group as assessed by means of angiography (p < 0.0001) and histologic quantification of blood vessels less than 80 microm diameter in local adipose tissue and capillaries per muscle fiber (p < 0.0002). AdVEGF treatment prevented a rise in femoral venous lactate femoral venous concentrations 1 hour after femoral artery ligation in control rats (p < 0.04). CONCLUSIONS: An adenovirus vector expressing VEGF complementary DNA is capable of stimulating an angiogenic response that protects against acute vascular occlusion in the setting of preexisting ischemia, suggesting that in vivo gene transfer of VEGF complementary DNA might be useful in prophylaxis of advancing arterial occlusive disease.

Adenoviridae↗
PubMed1998

The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro.

Trials of gene transfer for cystic fibrosis (CF) are currently underway. However, direct application to the airways may be impeded by the presence of airway secretions. We have therefore assessed the effect of CF sputum on the expression of the reporter gene beta-galactosidase complexed with the cationic liposome DC-Chol/DOPE in a number of cell lines in vitro. Transfection was markedly inhibited in the presence of sputum; the effect was concentration dependent and was only partially ameliorated by removal of sputum with phosphate-buffered saline (PBS) washing before gene transfer. However, treatment of the sputum-covered cells with recombinant human DNase (rhDNase, 50 micrograms/ml) but not with N-acetylcysteine, Nacystelyn, lysine (all 20 mM) or recombinant alginase (0.5 U/ml) significantly (P < 0.005) improved gene transfer. Adenovirus-mediated gene transfer efficiency in the presence of sputum was similarly inhibited, and again, treatment with rhDNase before transfection significantly improved gene transfer (P < 0.005). Transfection of Cos 7 cells in the presence of exogenous genomic DNA alone demonstrated similar inhibition to that observed with sputum and was also ameliorated by pre-treatment of DNA-covered cells with rhDNase. In a separate series of experiments performed in the absence of added sputum or genomic DNA, increasing concentrations of rhDNase resulted in a concentration-related decline in transfection efficiency. However, even at the highest concentration (500 micrograms/ml of rhDNase), transfection efficiency remained more than 50% of control. Thus, pre-treatment of CF airways with rhDNase may be appropriate before liposome or adenovirus-mediated gene therapy.

Adenoviridae↗
PubMed1998

Regional 'pro-drug' gene therapy: intravenous administration of an adenoviral vector expressing the E. coli cytosine deaminase gene and systemic administration of 5-fluorocytosine suppresses growth of hepatic metastasis of colon carcinoma.

Direct administration of an adenoviral vector expressing the cytosine deaminase gene (AdCMV.CD) to tumors of colon carcinoma cells, with concomitant systemic administration of 5-fluorocytosine (5FC), results in local production of 5-fluorouracil (5FU) and suppression of tumor growth. Based on the demonstration that in vivo adenovirus-mediated gene transfer to intrahepatic tumors is relatively inefficient compared with in vivo gene transfer to hepatocytes, we developed a 'regional' prodrug strategy using in vivo Ad-mediated CD gene transfer to normal liver, permitting hepatocytes to convert 5FC into 5FU to treat local metastasis effectively in a 'trans' fashion. To show that hepatocytes can generate and export sufficient 5FU to achieve this goal, primary rat hepatocytes were exposed to AdCMV.CD and 5FC. Evaluation of the supernatants by spectrophotometry and by HPLC demonstrated significant conversion of 5FC into 5FU. When supernatants of hepatocytes exposed to AdCMV.CD and 5FC were transferred to cultures of CT26 mouse colon carcinoma cells, the CT26 viability was reduced by 80%. To show that this regional AdCMV.CD/5FC prodrug strategy can suppress tumor growth in vivo, a model of metastatic colon carcinoma was established by injecting CT26 cells into the left lobe of the liver of syngeneic Balb/c mice. The next day, AdCMV.CD was transferred to hepatocytes by intravenous administration, and 5FC treatment was started the following day. Evaluation of tumor growth after 15 days showed marked suppression of tumor growth in AdCMV.CD- and 5FC- treated animals compared to control groups (P < 0.007). We conclude that primary hepatocytes are capable of converting 5FC into 5FU and exporting sufficient amounts of 5FU to the local milieu to suppress the growth of liver metastases of colon carcinoma cells.

Adenoviridae↗
PubMed1998

Adenovirus-mediated expression of melanoma antigen gp75 as immunotherapy for metastatic melanoma.

Melanocyte differentiation antigens, such as the brown locus protein gp75, are potential biological targets for immunotherapy. We investigated whether expression of the murine gp75 cDNA mediated by an adenovirus (Ad) vector could induce melanoma rejection using this model self antigen that usually induces tolerance, and whether Ad vector-directed production of interleukin-2 (IL2) might augment this response. To evaluate this approach, Ad vectors were constructed containing the murine gp75 cDNA (Ad.gp75) and the human IL2 cDNA (Ad.IL2). Efficacy was evaluated in C57BI/6 mice challenged i.v. with 10(5) B16 cells, using the number of lung metastases as the efficacy parameter. Naive control mice developed 175 +/- 12 metastases by day 14. Controls receiving intranasal Ad.IL2 1 day after B16 cell injection, intraperitoneal (i.p.) mitomycin-C-treated B16 cells +/- i.p. Ad.IL2 before B16 cell challenge and Ad.beta gal-treated mice had similar numbers of metastases as controls (P > 0.1). In marked contrast, preimmunization with intradermal Ad.gp75 provided dramatic reduction in the number of lung metastases (52 +/- 7, 29% of control). Addition of regional (intranasal delivery to the lung) Ad.IL2 to intradermal Ad.gp75 preimmunization 1 day following tumor challenge provided further protection (18 +/- 6, 10% of control). Depletion of CD4+ and CD8+ T-cell subsets effectively blocked the protective effect seen following immunization. Adoptive transfer of macrophage-depleted splenocytes from Ad.gp75-immunized mice similarly afforded significant protection against B16 tumor cell challenge. Further, serum obtained 21 days following Ad.gp75 immunization showed no detectable anti-gp75 antibody by immunoprecipitation. These results suggest that immunization with Ad.gp75 induces cellular immune responses that are capable of rejecting B16 melanoma in a host that is usually tolerant to gp75 antigen.

Adenoviridae↗
PubMed1998

Construction and characterization of hexon-chimeric adenoviruses: specification of adenovirus serotype.

This study has used the strategy of gene replacement to characterize the contribution of the adenovirus (Ad) capsid protein hexon to serotype definition. By replacing the Ad type 5 (Ad5) hexon gene with sequences from Ad2, we have changed the type specificity of the chimeric virus. The type-determining epitopes are primarily associated with loop 1 of hexon and, to a much lesser degree, with loop 2. In spite of the serotype distinctiveness of the chimeric hexon viruses, epitope similarity between the vectors resulted in a low level of cross-reactive neutralizing antibody, which in combination with activated cellular and innate arms of the immune system is sufficient to suppress gene transduction following readministration in vivo.

Adenoviridae Infections↗