Replication-deficient Recombinant Adenoviral Vector Research Articles

Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene.

To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were >15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P<0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micromol.L(-1) in 1000 M.O.I AdCEACD infected Lovo cells and 24.8+/-7.1 micromol.L(-1) in 100 M.O.I AdCMVCD infected Lovo cells, P<0.05, P<0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC(50) of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was >15000 and 214.5+/-31.3 micromol.L(-1), P<0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent. The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma.

Evaluation of endostatin antiangiogenesis gene therapy in vitro and in vivo.

Progressive growth and metastasis of solid tumors require angiogenesis, or the formation of new blood vessels. Endostatin is a 20-kDa carboxy-terminal fragment of collagen XVIII that has been shown to inhibit endothelial cell proliferation and tumor angiogenesis. Replication-deficient recombinant adenovirus (rAd) vectors were constructed, which encoded secreted forms of human and mouse endostatin (HECB and MECB, respectively), and, as a control, human alkaline phosphatase (APCB). Accumulation of endostatin was demonstrated in supernatants of cultured cells infected with the endostatin rAds. These supernatants disrupted tubule formation, inhibited migration and proliferation, and induced apoptosis in human dermal vascular endothelial cells or human vascular endothelial cells. Endostatin-containing supernatants had no effect on the proliferation of MidT2-1 mouse mammary tumor cells in vitro. A pharmacokinetic study of MECB in immunocompetent FVB mice demonstrated a 10-fold increase of serum endostatin concentrations 3 days after intravenous administration of 1x10(10) particles of this rAd (215-257 ng/mL compared to 12-38 ng/mL in control rAd-treated mice). Intravenous administration of MECB reduced b-FGF stimulated angiogenesis into Matrigel plugs by 38%. Intratumoral MECB inhibited growth of MidT2-1 syngeneic mammary tumors in FVB mice, but had minimal impact on the growth of MDA-MB-231 human breast tumors in SCID mice. Intravenous therapy with MECB also initially inhibited growth of MidT2-1 tumors, but this activity was subsequently blocked by induced anti-rAd antibodies. In summary, endostatin gene therapy effectively suppressed angiogenic processes in vitro and in vivo in several model systems.

Adenovirus-mediated expression of pig alpha(1, 3) galactosyltransferase reconstructs Gal alpha(1, 3) gal epitope on the surface of human tumor cells.

Gal alpha(1, 3) Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by alpha(1, 3) galactosyltransferase [alpha(1, 3) GT] enzyme on the cells of lower mammalian animals such as pigs and mice. Human has no gal epitope due to the inactivation of alpha(1, 3) GT gene but produces a large amount of antibodies (anti-Gal) which recognize Gal alpha(1, 3) Gal structures specifically. In this study, a replication-deficient recombinant adenoviral vector Ad5sGT containing pig alpha(1, 3) GT cDNA was constructed and characterized. Adenoviral vector-mediated transfer of pig alpha(1, 3) GT gene into human tumor cells such as malignant melanoma A375, stomach cancer SGC-7901, and lung cancer SPC-A-1 was reported for the first time. Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis, although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction. Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I (UEA I) lectins, Vicia villosa agglutinin (VVA), Arachis hypogaea agglutinin (PNA), and Glycine max agglutinin (SBA) to different degrees. In addition, no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.

Adenovirus-mediated p53 gene transfer to rat testis impairs spermatogenesis.

The tumor suppressor protein p53 participates in normal cell differentiation as well as induction of programmed cell death. The authors investigated the effect of p53 overexpression on spermatogenesis by transferring p53 gene into the rat testes. Replication-deficient recombinant adenovirus vectors were constructed to include cytomegalovirus (CMV) promoter driving wild-type p53 (Ad-CMV-p53) or beta-galactosidase (Ad-CMV-beta-gal). Virus was delivered to cells of the tubules by slow retrograde injection through the rete testis. At 0, 4, 7, and 14 days, testes were removed, weighed, and analyzed histopathologically, including immunohistochemistry for p53, Bcl-2, Bax, and interleukin-1beta converting enzyme (ICE). Testicular weight was decreased in Ad-CMV-p53 group at 14 days after injection, while no change occurred in phosphate-buffered saline-injected controls or Ad-CMV-beta-gal-infected testes. Beyond 4 days, cell degradation in tubules interfered with immunohistochemical observation in the Ad-CMV-p53 group. At 4 days, p53 was expressed mostly in spermatocytes. Bax showed greater expression in the p53 group than in the control or Ad-CMV-beta-gal group. ICE, expressed mostly in spermatids, was more abundant in the p53 group than in controls. Overall, p53 overexpression in the testis impaired spermatogenesis.

Over‐expression of p27kip1 induces growth arrest and apoptosis mediated by changes of pRb expression in lung cancer cell lines

p27kip1 is a cyclin-dependent kinase inhibitor which controls the G1 phase of the cell cycle in conjunction with pRb. p27 has been associated with cell-cycle arrest and apoptosis. In this study, we transferred the full-length human p27 cDNA using a replication-deficient recombinant adenoviral vector (Ax-p27) into lung cancer cell lines and evaluated the potential of this strategy for anti-cancer gene therapy. After infection with Ax-p27, the growth of H322, A549 and SQ-5 cells, which express pRb, was almost completely suppressed, though no such effect was found in H69 and Lu-135 cells, which do not express pRb. In addition, cell death from day 4 after infection with Ax-p27 was observed only in H322, A549 and SQ-5 cells but not in H69 and Lu-135 cells. The cell cycle of H322 cells treated with Ax-p27 became arrested at the G1 phase from day 1 to day 3 despite continued over-expression of p27. When we examined the changes in expression level of pRb and E2F-1, which play important roles in cell-cycle progression from G1 to S phase, down-regulation of pRb expression was detected in H322 cells 3 days after infection with Ax-p27. These data suggest that (i) the growth-inhibitory effect and induction of apoptosis by over-expression of p27 require expression of pRb and (ii) adenovirus-mediated p27 gene transfer may have promise as a novel strategy in cancer gene therapy. Int. J. Cancer 88:377–383, 2000. © 2000 Wiley-Liss, Inc.

Adenovirus-mediated tumor suppressor p53 gene therapy for anaplastic thyroid carcinoma in vitro and in vivo.

The present study was designed to evaluate the therapeutic efficacy of adenovirus-mediated wild-type (wt) tumor suppressor p53 expression in four human thyroid carcinoma cell lines harboring p53 mutations (ARO, FRO, NPA, and WRO) and normal human thyroid follicular cells with wt-p53 in vitro and in vivo. In vitro infection of replication-deficient recombinant adenovirus vector expressing wt-p53 led to a dose-dependent cell killing in both normal and carcinoma cells. In contrast, adenovirus expressing Escherichia coli beta-galactosidase showed little effect. The sensitivity to p53-mediated cell killing varied among the cells used. It was, at least partly, dependent on their adenovirus infectivity in carcinoma cells, whereas normal thyroid cells were relatively resistant to p53-mediated cell death despite its highest adenovirus infectivity. The mechanism of cell killing by wt-p53 was shown, by flow cytometric analysis, to be apoptosis. Furthermore, wt-p53 expression renders two out of four carcinoma cell lines (FRO and NPA) more sensitive to doxorubicin and one (FRO) to 5-fluorouracil, independent of treatment schedule. In vivo experiments, using FRO and NPA cells, showed that growth of sc tumors in nude mice was nearly completely inhibited by direct injection of adenovirus expressing wt-p53 [1 x 10(9) plaque-forming units/tumor]. This effect was augmented by its combination with doxorubicin treatment (4 mg/kg, thrice a week), which led to tumor regression. Our results therefore indicate that adenovirus-mediated wt-p53 gene introduction seems to be a potential clinical utility in gene therapy for anaplastic thyroid carcinomas, particularly when combined with chemotherapy.

Efficient Cre/loxP site-specific recombination in a HepG2 human liver cell line.

A worldwide shortage of donor livers is a limiting factor of the clinical application of hepatocyte transplantation (HTX). To resolve this issue, we focused on a reversible immortalization system that allows temporary expansion of primary hepatocyte populations by transfer of an oncogene that can be subsequently excised. As a preliminary test toward this goal, we examined the efficacy of Cre/loxP site-specific recombination in a transformed human liver cell line, HepG2. The present study utilized retroviral transfer of a prototypical immortalizing gene, simian virus 40 large T antigen (SV40Tag), flanked by a pair of loxP recombination targets and adenovirus-mediated Cre/loxP recombination. Here we report that complete elimination of the retroviral transferred oncogene was achieved by site-specific recombination using a replication-deficient recombinant adenovirus vector producing Cre recombinase (Ad-Cre).

Adenovirus-mediated gene transfer of basic fibroblast growth factor promotes the survival of primary-cultured rat neuronal cells.

We constructed two replication-deficient recombinant adenovirus vectors coding human basic fibroblast growth factor (bFGF), one with and one without the interleukin-2 (IL-2) secretory signal sequence and examined their neurotrophic effects on primary neuronal cells in vitro. The primary neuronal cells were successfully infected at a high efficiency with the adenovirus vectors. bFGF protein was detected in the culture medium of the neurons infected with both these vectors. The cells infected with the bFGF-expressing adenovirus containing the IL-2 signal sequence showed 2- to 10-fold higher levels of secretion levels than cells infected with the native bFGF-expressing adenovirus alone. Both bFGF-expressing vectors augmented the survival of primary neuronal cells in an in vitro culture, compared with a mock infection or control virus infection. Notably, the cells infected with the bFGF-expressing adenovirus containing the IL-2 signal sequence were markedly enhanced cell survival in the early phase of the culture, compared with the control cells and even those infected with the bFGF-expressing adenovirus without the IL-2 signal sequence. However, in the late phase of neuronal culture, neither viral vector could support the cell survival. In contrast the co-infection of the bFGF-expressing vector with a Bcl-xL-expressing vector was extremely effective on neuronal survival.

Immunological analysis of the tegument phosphoprotein ppUL83 of human cytomegalovirus

Immunological properties of the tegument phosphoprotein, ppUL83, of human cytomegalovirus (HCMV), expressed using a replication deficient recombinant adenovirus vector (RAd83) are described. The initial characterisation of this protein was carried out by immunofluorescence (IF), immunoprecipitation (RIP) and immunoblotting using nine mouse monoclonal antibodies (Mabs) directed against five linear and four conformational epitopes of ppUL83. The reactivity of the recombinant protein with the Mabs was similar to that observed with native ppUL83, although, the kinetics of its expression was in agreement with expression derived from the HCMV major immediate early promoter (MIEP). The recombinant antigen was used successfully in an Enzyme Immunoassay (EIA) for the detection of IgG class antibodies in 171 sequential sera taken from 21 heart transplant recipients. Comparison of HCMV-infected and RAd83-infected cell extracts in this experiment showed that recombinant antigen could substitute whole virus extracts as a single well-characterised protein in EIA. Serum IgG avidity measurements, using the recombinant ppUL83, differentiated between primary and past HCMV infections in the population studied.

Over-expression of p27kip1 induces growth arrest and apoptosis mediated by changes of pRb expression in lung cancer cell lines

p27kip1 is a cyclin-dependent kinase inhibitor which controls the G1 phase of the cell cycle in conjunction with pRb. p27 has been associated with cell-cycle arrest and apoptosis. In this study, we transferred the full-length human p27 cDNA using a replication-deficient recombinant adenoviral vector (Ax-p27) into lung cancer cell lines and evaluated the potential of this strategy for anti-cancer gene therapy. After infection with Ax-p27, the growth of H322, A549 and SQ-5 cells, which express pRb, was almost completely suppressed, though no such effect was found in H69 and Lu-135 cells, which do not express pRb. In addition, cell death from day 4 after infection with Ax-p27 was observed only in H322, A549 and SQ-5 cells but not in H69 and Lu-135 cells. The cell cycle of H322 cells treated with Ax-p27 became arrested at the G1 phase from day 1 to day 3 despite continued over-expression of p27. When we examined the changes in expression level of pRb and E2F-1, which play important roles in cell-cycle progression from G1 to S phase, down-regulation of pRb expression was detected in H322 cells 3 days after infection with Ax-p27. These data suggest that (i) the growth-inhibitory effect and induction of apoptosis by over-expression of p27 require expression of pRb and (ii) adenovirus-mediated p27 gene transfer may have promise as a novel strategy in cancer gene therapy.

CYTOTOXICITY OF ADENOVIRAL-MEDIATED CYTOSINE DEAMINASE PLUS 5-FLUOROCYTOSINE GENE THERAPY IS SUPERIOR TO THYMIDINE KINASE PLUS ACYCLOVIR IN A HUMAN RENAL CELL CARCINOMA MODEL

Purpose An estimated 11,600 Americans will die of renal cell carcinoma in 1998. The lack of effective chemotherapy or radiotherapy requires the investigation of novel treatment modalities. We compared two forms of toxic gene therapy, cytosine deaminase (CD) plus 5-fluorocytosine (5-FC) and thymidine kinase (TK) plus acyclovir (ACV), in pre-clinical models of human renal cell carcinoma. Materials and Methods Replication-deficient recombinant adenoviral vectors containing the Rous sarcoma virus promoter driving CD (Ad-RSV-CD) or TK (Ad-RSV-TK) gene expression were constructed and tested for in vitro cell-killing assays at various viral multiplicity of infection (MOI) and in vivo for growth inhibition of a human renal cell carcinoma, SK-RC-29 models. Subcutaneous tumors of SK-RC-29 were examined by electron microscopy for presence of intercellular gap junctions. Levels of expression of the gap junctional associated connexin 43 protein in SK-RC-29, 31, 38, 42, 52 human RCC cell lines were examined by western immunoblotting. Results In vitro cell-killing assay comparing Ad-RSV-CD/5F-C and Ad-RSV-TK/ACV at a wide range of MOI (2.5 to 20) revealed superior cell-kill by Ad-RSV-CD/5-FC over Ad-RSV-TK/ACV. Consistent with these results, we observed that Ad-RSV-CD/5-FC but not Ad-RSV-TK/ACV demonstrated a significant in vivo tumor growth inhibition. These results are corroborated by the lack of gap junctions in SK-RC-29 subcutaneous tumors by the electron microscopy and the absence of connexin-43 in all five human RCC cell lines by western immunoblotting. Conclusion We have demonstrated in this study that Ad-RSV-CD/5-FC is superior to Ad-RSV-TK/ACV for the treatment of human RCC in cell culture and animal models. The results are supported by the lack of gap junctional communication between RCC cells assessed by connexin-43 expression.

Adenovirus-mediated gene transfer of Bcl-xL prevents cell death in primary neuronal culture of the rat

Bcl-xL is a Bcl-2-related gene that regulates programmed cell death in a bcl-2-independent fashion. It is expressed in tissues containing long-surviving postmitotic cells, such as neurons in adult brains. To investigate the possibility of gene therapy for transferring this anti-apoptotic gene into the neuron for the treatment of vascular occlusive or neurodegenerative diseases, we examined the effect of a replication-deficient recombinant adenovirus vector coding human Bcl-xL gene on the augmentation of the survival of primarily-cultured rat neuronal cells in vitro. Immunoblot analysis revealed that primarily-cultured neuronal cells were successfully infected and transferred with this gene by recombinant adenovirus vector with high transduction efficiency. Bcl-xL gene transfer to the primarily-cultured neurons could prevent these cells from cell death.

Adenovirus-mediated gene transfer of a truncated form of fibroblast growth factor receptor inhibits growth of glioma cells both in vitro and in vivo.

Basic fibroblast growth factor (FGF-2) and high affinity FGF receptor (FGFR) have been detected in the nucleus as well as the cytoplasm of many human gliomas, and are known to stimulate cellular proliferation and angiogenesis in the tumors. To investigate the effects of inactivation of FGFR on the growth of malignant gliomas, we constructed a replication-deficient recombinant adenovirus vector encoding a truncated form of chicken FGFR1 (AxCA delta FR). AxCA delta FR-infected cells were confirmed to express truncated FGFR protein by immunoblotting and FGF-2-dependent clonogenicity of NIH3T3 cells was suppressed by infection with this virus vector. Then human malignant glioma cell lines U-251MG and T98G, both of which have been reported to express FGF-2 and FGFR, were infected with AxCA delta FR. These infected cells showed nuclear as well as cytoplasmic expression of a truncated FGFR protein. Proliferation rate and the ability to form colonies in soft agar of the cells infected with this virus vector were significantly suppressed compared with those of uninfected and lacZ-expressing adenovirus-infected cells. Moreover, intratumoral injection of AxCA delta FR significantly suppressed the subcutaneous tumor growth of the glioma cells in nude mice. We concluded that inactivation of the cytoplasmic and nuclear FGFR using this truncated FGFR-expressing adenovirus vector can inhibit the growth of malignant gliomas both in vitro and in vivo.

Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery after experimental subarachnoid hemorrhage.

Gene transfer with recombinant viral vectors encoding vasodilator proteins may be useful in therapy of cerebral vasospasm after subarachnoid hemorrhage (SAH). Relaxations mediated by nitric oxide are impaired in cerebral arteries affected by SAH. The present study was designed to determine the effect of SAH on the efficiency of ex vivo adenovirus-mediated gene transfer to canine basilar arteries and to examine whether expression of recombinant endothelial nitric oxide synthase (eNOS) gene may have functional effects on vasomotor reactivity of spastic arteries affected by SAH. Replication-deficient recombinant adenovirus vectors encoding bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVbeta-Gal) genes were used for ex vivo gene transfer. Rings of basilar arteries obtained from control dogs and dogs exposed to SAH were incubated with the vectors in minimum essential medium. Twenty-four hours after gene transfer, expression and function of the recombinant genes were evaluated by (1) histochemical or immunohistochemical staining, (2) beta-galactosidase protein measurement, and (3) isometric tension recording. Transduction with AdCMVbeta-Gal and AdCMVeNOS resulted in the expression of recombinant beta-galactosidase and eNOS proteins mostly in the vascular adventitia. The expression of beta-galactosidase protein was approximately 2-fold higher in SAH arteries than in normal arteries. Endothelium-dependent relaxations caused by bradykinin and substance P were suppressed in SAH arteries. The relaxations to bradykinin were significantly augmented in both normal and SAH arteries after AdCMVeNOS transduction but not after AdCMVbeta-Gal transduction. The relaxations to substance P were augmented by AdCMVeNOS transduction only in normal arteries. Bradykinin and substance P caused relaxations even in endothelium-denuded arteries, when the vessels were transduced with AdCMVeNOS. These endothelium-independent (adventitia-dependent) relaxations to bradykinin observed after AdCMVeNOS transduction were similar between normal and SAH arteries, whereas those to substance P were significantly reduced in SAH arteries compared with normal arteries. These results suggest that expression of recombinant proteins after adenovirus-mediated gene transfer may be enhanced in cerebral arteries affected by SAH and that successful eNOS gene transfer to spastic arteries can at least partly restore the impaired nitric oxide-mediated relaxations through local (adventitial) production of nitric oxide.

GENE THERAPY FOR COLON CANCER

The enormous number of newly diagnosed cases of colorectal cancer that occur each year and the lack of agents that are highly effective for all patients underscore the need for novel approaches to combating the disease. Gene therapy as a developing treatment modality is already well established, with a number of trials ongoing and a vast range of other approaches being assessed in animal and cell culture experiments. In this brief review, we have discussed five gene therapy trials in colon carcinoma that are ongoing or in the approval process in the United States. The gene therapy approaches being employed can be divided into three major categories: (1) enzyme/prodrug systems (HSVtk/ganciclovir; CD/5-fluorocytosine); (2) tumor suppressor gene replacement therapy with wild-type p53; and (3) immune-gene therapy which is based on cytokine or tumor antigen expression to induce tumor immunity (e.g., CEA). Replication-deficient recombinant adenoviral vectors are predominantly used for colon cancer gene therapy, because they can be produced at high titer and they readily infect a number of different cell types. One trial uses polynucleotide therapy for antitumor immunization with intramuscular injection. All of these studies are phase I trials, principally designed to evaluate safety, but they will also provide data on gene delivery. Some trials may provide some insight into potential therapeutic effects. We have alluded to some of the concerns on toxicity related to the use of adenovirus, risks and side effects from transgenes, lack of tumor-specificity of transgene expression, and potential problems with efficient gene delivery to solid tumors. The clinical trials, however, will provide insight that will inform design of future studies with respect to dose, form, and frequency of administration, as well as to the value of biologic and clinical endpoints. The molecular analysis of the fundamental basis of colon cancer has moved at a remarkable pace and that progress seems set to continue. Thus, the basic foundations for gene therapy are undoubtedly in place: a clinical need; growing understanding of basic tumor biology; and ever-improving delivery systems. The field is at a very early stage in its evolution, and one concern is that the considerable hurdles that must be overcome are seen as examples of the failure of cancer gene therapy; however, we believe these challenges will be overcome. The authors also believe that colon cancer gene therapy is likely to take new directions, such as use as adjuvant to radical surgery, rather than attempts to treat end-stage disease when the liver is replaced by metastases. Other new directions might include prophylactic gene-based immunization against a panel of well-characterized tumor antigens, at least for persons shown to be at high risk of colon cancer because of genetic or other predisposition. A marriage between gene therapy approaches and conventional anticancer treatments such as radiotherapy and chemotherapy also seems likely. There is already evidence of this move with demonstration of synergism between p53 replacement and radiotherapy and chemotherapy. It is also likely that therapies will be developed that combine elements from the cancer gene therapies discussed previously, namely, suicide gene transfer, immune modulation, and modulation of defective cancer genes. Perhaps one of the main concerns is not that researchers in cancer gene therapy want to walk before they can run, but that the public and government agencies believe they can. The next 10 years will be an interesting time in the development of novel treatments against colon cancer.

Repetitive adenovirus administration to the parotid gland: role of immunological barriers and induction of oral tolerance.

This study assessed the mucosal and systemic immune responses following repetitive adenoviral vector instillation to the parotid glands. Also, we investigated the feasibility of oral tolerance induction as a rational strategy to overcome the immunological reactions. The replication-deficient recombinant adenovirus vector AdCMVCAT was instilled into rat parotid glands. Chloramphenicol acetyltransferase (CAT) activity in the parotid was observed after a first or second AdCMVCAT infection, but not after a third vector administration. ELISA assays showed increased anti-adenovirus immunoglobulin G (IgG) and IgM in serum, and also anti-adenovirus IgA in gland extracts and saliva after virus administration. The results of in vivo neutralization experiments demonstrated that salivary IgA and IgM prevented reinfection of the parotids with adenoviral vectors. Subsequently, studies were conducted to induce tolerance to adenovirus by peroral feedings of ultraviolet (UV)-inactivated virus before gene administration to the parotid glands. Between 3 and 13 doses of virus were fed to rats. Final parotid gene expression was dependent on the number of viral feedings and the amount fed. Tolerized animals showed prolonged and heightened gene expression in the salivary glands compared to control animals and displayed gene expression even after three administrations of vector. Mononuclear cells from the spleens of these animals showed reduced proliferation following adenovirus stimulation. This same cell population was depleted of CD8+ T cells and found to produce less interferon-gamma (IFN-gamma) after virus challenge. This profile indicates the down regulation of Th1 cell-mediated responses. These results indicate that oral tolerance induction is a potentially useful adjunct to virus-based gene therapy.

Adenovirus-mediated gene expression in isolated rat pancreatic acini and individual pancreatic acinar cells.

In this study we have examined the feasibility of using replication-deficient recombinant adenoviral vectors to transfer and express genes in pancreatic acinar cells in vitro. We infected primary cultures of both isolated pancreatic acini and individual acinar cells with a recombinant adenovirus containing the coding sequence for beta-galactosidase. Our data demonstrate that recombinant adenoviruses readily infect pancreatic acinar cells in vitro. Close to 100% infection and maximal beta-galactosidase expression were obtained, when acini or acinar cells were infected with 5x10(6) or 10(6) plaque-forming units (pfu) of virus per millitre of acini or acinar cell suspension, respectively. Examination of the time-course of beta-galactosidase expression showed that there was a lag of approximately 6 h before beta-galactosidase levels increased. Thereafter beta-galactosidase expression increased rapidly. By 20 h post-infection beta-galactosidase activity had increased from undetectable levels to 2.5-3.0 units/mg of cellular protein. Acini/acinar cells maintained a robust secretory response after adenoviral infection. The cholecystokinin-octapeptide (CCK8) dose/response curves for amylase secretion for acini and acinar cells infected with 5x10(5) and 1x10(5) pfu/ml of virus, respectively, were biphasic, with maximal amylase secretion being stimulated by 1 nM CCK8. In addition, the dose/response curves were identical to those obtained from control, sham-infected, acini/acinar cells. Our findings indicate that replication-deficient recombinant adenoviral vectors will be excellent tools to transfer and express genes in isolated pancreatic acini or acinar cells.

In utero cardiac gene transfer via intraplacental delivery of recombinant adenovirus.

The relationship among the maternal, placental, and uniquely shunted embryonic circulation was explored to provide access to the embryonic cardiovascular system in utero. Manipulation of gene expression in the developing heart would be particularly useful for studying the effects of altered gene expression on cardiac development and in the etiology of congenital cardiac anomalies. Dye studies demonstrated that intraplacental injection allows direct access to the embryonic cardiac and systemic circulation. To evaluate the efficacy of cardiac gene transfer using this approach, replication-deficient recombinant adenoviral vectors encoding luciferase or beta-galactosidase as reporter genes were injected intraplacentally into embryonic day (E)12.5 murine embryos, an age at which the mass of the heart was observed to be large compared with other organs. Embryos were assayed for transgene expression at E15.5 and at birth. Survival rates at these times were similar among vector-injected and control groups. At E15.5 and at birth, luciferase activity within the heart was 9- and 23-fold higher, respectively, than in the remainder of the embryo, although levels of expression were generally lower at birth than during embryonic life. Beta-galactosidase expression was observed within all regions of the embryonic heart and was localized to approximately 15% of atrial and ventricular cells. Intraplacental delivery of adenovirus at embryonic day 12.5 results in somatic gene transfer to the murine embryonic heart, which persists at least until birth. The combination of intraplacental injection to directly access the fetal coronary circulation and injection at E12.5 when the mass of the heart is large compared with other organs results in transgene expression in cardiac cells. Intraplacental injections early in embryonic life may thus be useful to study the effects of temporal manipulation of gene expression on cardiac development and disease.

Adenovirus-mediated gene transfer of fibroblast growth factor-1: angiogenesis and tumorigenicity in nude mice.

Gene transfer of angiogenic growth factors with replication-deficient recombinant adenovirus (Ad) vectors may provide a new approach to the treatment of ischemic diseases. To determine if Ad-infected cells could stimulate angiogenesis in vivo and to assess the tumorigenicity of cells infected with these vectors, NIH3T3 fibroblasts infected with Ad vectors coding for human acidic fibroblast growth factor (aFGF-1) were used in angiogenic and tumorigenic assays. Infected cells induced a strong angiogenic response in vivo, while cells infected with control virus did not. Stable 3T3 transfectants expressing the FGF-1 gene were also highly angiogenic and exhibited growth in soft agar, while Ad-infected cells did not. Ad-infected cells grew transiently in nude mice, whereas 3T3 transfectants formed large tumors which grew exponentially. Extrapolation of cell dose-response curves showed that a minimum of 1.5 x 10(4) infected cells were required for transient tumor cell growth in vivo. Ad-infected cells cultured in vitro for 30 days lost their invasive phenotype and the ability for transient cell growth in nude mice. Thus, phenotypic changes induced by Ad-mediated gene transfer of FGF-1 are transient both in vitro and in vivo, suggesting that these Ad vectors do not have tumorigenic potential. Stimulation of angiogenesis by Ad-infected cells may be useful for the evaluation of anti-angiogenic and anti-tumor agents.

Endothelial Nitric Oxide Synthase Gene Transfer Inhibits Platelet-Derived Growth Factor-BB Stimulated Focal Adhesion Kinase and Paxillin Phosphorylation in Vascular Smooth Muscle Cells

Stimulation of smooth muscle (VSM) cells of guinea pig coronary artery by platelet-derived growth factor (PDGF)-BB retards paxillin mobility (mobility shift) in SDS-PAGE in a time-dependent manner. This mobility shift may be due to tyrosine phosphorylation of paxillin. eNOS gene transfer by replication deficient recombinant adenovirus vector AD5/RSVeNOS in VSM cells inhibited PDGF-BB-stimulated mobility shift and tyrosine phosphorylation of paxillin. Concomitantly, tyrosine phosphorylation of focal adhesion kinase (FAK) was also inhibited. The inhibition of paxillin and FAK tyrosine phosphorylation did not affect stress fiber and focal adhesion formation. Considering the importance of FAK and paxillin in cell migration and proliferation, these results suggest that the FAK-paxillin pathway is a target for NO action to inhibit VSM cell migration and proliferation.