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Biomedical subjects

M Perricaudet

Publications and source records attributed to M Perricaudet.

At least 55 records · Page 3Linked to original sources
PubMed1997

Mechanism of adenovirus improvement of cationic liposome-mediated gene transfer.

Substantial effort has been focused on the development of highly efficient gene transfer strategies. Although viral and non-viral methods have been elaborated, mechanisms of gene delivery are still poorly understood. We exploited our recent observation that replication-deficient type 5 adenovirus dramatically enhances lipofectAMINE-mediated gene transfer (lipoadenofection) in differentiated cells to elucidate the mechanism of adenovirus action in this process. Heat-induced denaturation of viral capsid abolishes adenovirus action whereas inactivation of viral genome by short treatment with UV has no effect. Electron microscopic observations reveal the formation of a complex containing adenovirus and lipofectAMINE which probably carries DNA into cells via endocytosis. Anti-adenovirus antiserum or monoclonal anti-alpha(v)beta3 integrin antibody inhibits lipoadenofection, at least partially. Neutralization of endosomal compartments with chloroquine, ammonium chloride or monensin does not prevent adenovirus improvement of gene transfer. Hence, adenovirus-lipofectAMINE-DNA complexes in which viral particles are each encompassed by three lipid layers, penetrate cells via an endocytic pathway involving probably the adenovirus receptor and alpha(v)beta3 integrin. The resulting efficient transfer and expression of plasmid DNA proceeds from a mechanism in which adenoviral endosomolytic activity appears to be required while viral genome is not essential.

Adenoviridae↗
PubMed1997

Phenotypic alteration of astrocytes induced by ciliary neurotrophic factor in the intact adult brain, As revealed by adenovirus-mediated gene transfer.

Synthesis of the ciliary neurotrophic factor (CNTF) and its specific receptor (CNTFRalpha) is widespread in the intact CNS, but potential biological roles for this system remain elusive. Contradictory results have been obtained concerning a possible effect on the morphological and biochemical phenotype of astrocytes. To reassess this question, we have taken advantage of adenovirus-mediated gene transfer into the rat brain to obtain the local release of CNTF. Stereotaxic administration of CNTF recombinant adenovirus vectors into the striatum led to phenotypic changes in astrocytes located in regions that were related axonally to striatal neurons at the injection site. Astrocytes appeared hypertrophied and displayed an increase in both GFAP and CNTF immunoreactivity. This response was observed up to 5 weeks after injection, the longest time studied. It was not observed after the administration of a control vector. The methodology used in the present study, allowing us to analyze the effect of the factor in areas remote from the injection site, has provided conclusive evidence that CNTF affects the astroglial phenotype in the intact CNS. The characteristics of these effects may explain why contradictory results have been obtained previously, because this signaling system seems to have a low efficiency and therefore requires a high local concentration of the factor close to the target cells. One might speculate as to the involvement of a CNTF astroglio-astroglial signaling system in the organized response of a population of astrocytes to changes in CNS homeostasis detected locally, even by a single cell.

Adenoviridae↗
PubMed1997

Reduction of restenosis after angioplasty in an atheromatous rabbit model by suicide gene therapy.

BACKGROUND: Gene delivery of the thymidine kinase (tk) gene combined with ganciclovir (GCV) limits intimal hyperplasia after abrasion of normal arteries. However, the low efficiency of adenoviral-mediated gene transfer to atherosclerotic arteries has raised concerns about the applicability of this strategy to the prevention of restenosis. METHODS AND RESULTS: A replication-defective adenoviral vector expressing tk (Ad-RSVtk) demonstrated selective toxicity toward GCV-treated arterial smooth muscle cells, with oligonucleolytic cleavage suggesting apoptosis. In vivo, after demonstration of tk expression after Ad-RSVtk delivery, the combination of Ad-RSVtk followed by GCV was tested in a rabbit model of angioplasty of atheromatous iliac arteries. Angioplasty (8 atm, 20 minutes) was performed by use of a hydrogel balloon coated with Ad-RSVtk (4x10(9) plaque forming units). GCV was infused (25 mg.kg(-1) I.V. BID) from days 2 through 7 after angioplasty in 8 of 12 rabbits. Four weeks later, morphometric analysis demonstrated a reduced intima-to-media ratio in the group receiving combination therapy compared with Ad-RSVtk alone (3.0+/-1.2 versus 5.2+/-0.5, P<.018). GCV per se had no effect on intimal hyperplasia after arterial injury. CONCLUSIONS: In vitro, Ad-RSVtk demonstrates selective toxicity toward GCV-treated arterial smooth muscle cells involving apoptosis. In vivo, GCV conditions reduction of neointimal formation after percutaneous delivery of Ad-RSVtk during angioplasty of atheromatous arteries.

Angioplasty, Balloon↗
PubMed1997

Enhancement of humoral immunity to SIVenv following simultaneous inoculation of mice by three recombinant adenoviruses encoding SIVenv/poliovirus chimeras, Tat and Rev.

A means of inducing gene expression by simultaneous infection with three recombinant adenoviruses (Ad) is described. The simian immunodeficiency virus (SIV) envelope-coding region was placed under the control of the human immunodeficiency virus type 1 (HIV-1) Tat and Rev proteins provided in trans by distinct Ad vectors (Ad-tat; Ad-rev). Coinfection of cells with the three recombinant adenoviruses led to induction of high levels of SIV env mRNA and protein synthesis, while inoculation of mice elicited anti-Env antibodies. Insertion of the poliovirus VP1 neutralization epitope (C3) in the V1 hypervariable region of SIV envelope not only proved to be highly immunogenic for the poliovirus epitope but also enhanced the kinetics of anti-SIV Env antibody production. By contrast, insertion in V4 elicited no anti-C3 response and only normal anti-Env responses.

Adenoviridae↗
PubMed1997

IL-2 gene delivery within an established murine tumor causes its regression without proliferation of preexisting antitumor-specific CTL.

Regression of P815 tumors established on naive syngeneic mice can be obtained by the intratumoral injection of a single dose of an adenoviral vector expressing the IL-2 gene (Ad.IL2). Injection triggers local IL-2 production for at least 10 days. We measured a number of immunologic parameters in situ following intratumoral Ad.IL2 treatment. We also analyzed the situation of regression obtained upon challenge with P815 cells of mice previously immunized against the tumor and compared both systems. While IFN-gamma messenger RNA expression was found to be elevated in both situations of tumor regression, the level of infiltration by tumor-specific CTL was different. A small amount of tumor-specific CD8+ T cells were present in growing, untreated tumors. Such cells are found in much larger numbers in tumors rejected upon challenge, consistent with a CTL-mediated rejection. In contrast, they were found not to proliferate following Ad.IL2 injection. The latter caused an increased infiltration of a polyclonal, presumably nonspecific, T cell population. These results suggest that the initial regression of established P815 tumors following Ad.IL2 treatment in vivo is mostly due to nonspecific effectors.

Animals↗
PubMed1997

Recombinational construction in Escherichia coli of infectious adenoviral genomes.

A two-step gene replacement procedure was developed that generates infectious adenoviral genomes through homologous recombination in Escherichia coli. As a prerequisite, a human adenovirus serotype 5 (Ad5)-derived genome was first introduced as a PacI restriction fragment into an incP-derived replicon which, in contrast to ColE1-derivatives (e.g., pBR322 or pUC plasmids), is functional in a polA mutant of E. coli. Any modification can be introduced at will following two consecutive homologous recombinations between the incP/Ad5 replicon and the ColE1 plasmid. The overall procedure requires only the in vitro engineering of the ColE1-derivative by flanking the desired modification with small stretches of identical sequences. In the first step, a cointegrate between the tetracycline-resistant incP/Ad5 replicon and the kanamycin-resistant ColE1-derivative is selected by growing the polA host in the presence of both antibiotics. Resolution of this cointegrate is further selected in sucrose growth conditions due to the loss of a conditional suicide marker (the sacB gene of Bacillus subtilis) present in the ColE1 plasmid, leading to unmodified and modified incP/Ad5 replicons that can be differentiated upon restriction analysis. Consecutive rounds of this two-step cloning procedure allowed the introduction of multiple independent modifications within the virus genome, with no requirement for an intermediate virus. The potential of this procedure is demonstrated by the recovery of several E1E3E4-deleted adenoviruses following transfection of the corresponding E. coli-derived genomes in IGRP2 cells.

Adenovirus E1 Proteins↗
PubMed1997

Prevention of bleomycin-induced pulmonary fibrosis after adenovirus-mediated transfer of the bacterial bleomycin resistance gene.

A serious limitation in the use of the DNA-cleaving, antitumoral-antibiotic, bleomycin during chemotherapy is pulmonary toxicity. Lung injury induced by bleomycin is characterized by an increased deposition of interstitial extracellular matrix proteins in the alveolar wall that compromises respiratory function. Several drugs have been tested in animal models to prevent the pulmonary toxicity of bleomycin, but have not led to a useful clinical treatment because of their adverse effects on other tissues. We have shown that transgenic mice expressing Streptoalloteichus hindustanus (Sh) ble bleomycin resistance protein in pulmonary epithelial cells in the lungs are protected against bleomycin-induced toxicity in lungs. In the present study, we used intranasal administration by adenovirus-mediated gene transfer of the bleomycin resistance Sh ble gene to mouse lung for prevention of bleomycin-induced pulmonary fibrosis. We constructed recombinant adenoviruses Ad.CMVble and Ad.RSVble harboring the bleomycin resistance Sh ble gene under the control of the cytomegalovirus early promoter and the Rous sarcoma virus early promoter, respectively. Transgene expression was detected in epithelia of conducting airways and alveolar septa by immunostaining with a rabbit polyclonal antibody directed against the bleomycin resistance protein and persisted for the duration of drug treatment; i.e., up to 17 d. No toxic effect was seen in adenovirus-treated mice. Pretreatment of mice with Ad.CMVble or Ad.RSVble completely prevented collagen deposition 42-133 d after bleomycin treatment, as measured by lung OH-proline content. Histologic studies indicated that there was little or no lung injury in the adenovirus/bleomycin-treated mice compared with the bleomycin-treated mice. These observations may lead to new approaches for the prevention of bleomycin-induced pulmonary fibrosis.

Acetyltransferases↗
PubMed1997

Expression from cardiomyocyte-specific promoter after adenovirus-mediated gene transfer in vitro and in vivo.

Adenoviruses are very attractive vectors for gene transfer into the cardiac muscle; however, their promiscuous tissue tropism, leading to an ectopic expression of the transgene, is a considerable practical limitation. To restrict expression of a reporter gene in cultured cardiomyocytes and in the heart of the rat, we have constructed a recombinant adenovirus (Ad-MLC2 beta gal) containing the beta-galactosidase gene under the control of the rat ventricle-specific cardiac myosin light chain 2 (MLC-2v) promoter. We show in this work that the MLC-2v promoter inside the adenoviral genome retains its cardiac specificity in vitro in cultured cardiomyocytes as well as in vivo in the animal heart. Northern blot studies after Ad-MLC2 beta gal infection show significant transcription only in cells derived from the cardiac muscle and not from the skeletal muscle. Quantitative analysis of the beta-galactosidase activity in a number of cell lines also confirms this result. The level of beta-galactosidase expression in rat neonatal cardiomyocytes infected with Ad-MLC2 beta gal is 8% of that found when primary cells are infected with Ad-RSV beta gal (containing a beta-galactosidase gene under the control of the Rous sarcoma virus promoter). The cardiomyocytes-specific expression is also found after injection of Ad-MLC2 beta gal directly into the rat myocardium, although the viral genome can be detected by polymerase chain reaction (PCR) in other tissues. Lack of expression after direct injection into liver and skeletal muscle confirms these results. The use of a tissue-specific promoter is a first step to restrict transgene expression to a particular cell type of the targeted tissue.

Animals↗
PubMed1997

Differential toxicity of ganciclovir for rat neurons and astrocytes in primary culture following adenovirus-mediated transfer of the HSVtk gene.

The toxicity of the suicide HSVtk gene approach is known to be targeted to DNA synthesis and, consequently, to dividing cells. This system is therefore useful for the treatment of brain tumors which contain dividing cells surrounded by a quiescent normal tissue. Adenoviruses are efficient vectors for the transfer of the HSVtk gene into the tumor but this can lead to the transduction of quiescent cells. In this study, we focused on the toxicity of the HSVtk/ganciclovir treatment for the two main cell types of the normal brain: astrocytes and neurons. Astrocytes and neurons in primary culture were infected by an adenoviral vector bearing the HSVtk gene (Ad.tk) and cells were exposed to different concentrations of ganclclovir. After 5 days of treatment, an MTT test measured a dramatic decrease in cell viability for treated astrocytes while a small decrease in cell viability was observed for neurons treated in the same experimental conditions. The differential toxicity of the HSVtk/ganciclovir treatment was also observed in cocultures of astrocytes and neurons: an immunocytochemical analysis of the treated cells showed major morphological modifications for astrocytes but not for neurons. Furthermore, our data suggest that a bystander effect is able to kill all the astrocytes while neurons from the same culture remain unaffected.

Adenoviridae↗
PubMed1997

Improved efficiency of arterial gene transfer by use of poloxamer 407 as a vehicle for adenoviral vectors.

Improvement in the efficiency of adenovirus-mediated arterial gene transfer may augment the utility of cardiovascular gene therapy. In vitro studies suggest that poloxamer 407 enhances transfection efficiency of adenoviral vectors in vascular smooth muscle cells. The aim of the present study was to investigate whether poloxamer 407 facilitates adenovirus-mediated arterial transfection in vivo as well. Gene transfer was performed in balloon-injured rat carotid arteries using E1- adenoviral vectors diluted in either poloxamer 407 or phosphate buffered saline (PBS). Transfection efficiency was significantly higher in rats transfected using a nuclear beta-galactosidase expressing adenovector diluted in poloxamer 407 versus PBS (morphometry, 13.2 +/- 1.3% versus 4.1 +/- 0.4% transfected medial cells, P = 0.0001; chemiluminescence; 1.4 +/- 0.2 versus 0.4 +/- 0.2 mU beta-galactosidase/mg protein, P = 0.004). Moreover, in the presence of poloxamer 407, it was possible to reduce the incubation time of adenoviral vectors from 20 to 10 min without compromising transfection efficiency. Poloxamer 407 did not evoke specific tissue toxicity. Site-specificity of arterial gene transfer, assessed by PCR, was not altered by administration of poloxamer 407. These findings suggest that poloxamer 407 may be useful to improve the efficiency of adenovirus-mediated arterial gene transfer.

Adenoviridae↗
PubMed1997

Adenovirus-mediated gene transfer of a human IL-6 antagonist.

IL-6 is a pleiotropic cytokine and plays a major role in inflammation and in the immune response. Altered serum levels of IL-6 have been described in several pathologies such as myeloma, EBV-lymphoma and chronic autoimmune disease. Here we report data on the utilization of a hIL-6 receptor superantagonist with a gene therapy approach. The superantagonist used in this work possesses very high affinity for the hIL-6 receptor, and is therefore an excellent candidate for the treatment of IL-6-dependent diseases. To obtain an efficient in vivo delivery method, we constructed a recombinant adenovirus expressing the IL-6 receptor superantagonist by inserting the cDNA, controlled by the RSV promoter, into a first generation replication-incompetent adenoviral vector. Recombinant virus allowed correct expression of the transgene in vitro. Supernatants of infected cells specifically inhibited IL-6-induced transcriptional activation in hepatoma cells and blocked the IL-6-dependent proliferation of human myeloma cells. After intravenous injection of the recombinant virus into mice, nanomolar amounts of antagonist were produced in the serum, and these were able completely to inhibit IL-6 bioactivity. Gene transfer of such an antagonist offers a practical means of imposing long-term blockade of IL-6 activity in vivo for investigational and therapeutic purposes.

Adenoviridae↗
PubMed1997

Advances in adenoviral vectors: from genetic engineering to their biology.

Ad2 and Ad5 belong to a group of human cytolytic viruses that target the respiratory airways for reproduction, whereas latent infections establish within other tissues. Signals therefore exist that control this dichotomic process in different cell types, perhaps including cis and/or trans elements of viral origin. Since 1993, Ad2- and Ad5-based adenoviruses lacking all or part of the E1 regulatory region have been undergoing evaluation in phase I trials that target cancer and cystic fibrosis. These viruses are extremely attenuated and actually do not reproduce in most human cells. However, they retain most of the virus genetic program and often promote a significant cytotoxicity after infection, emphasizing the need to further cripple the virus biology to extend the duration of transgene expression, if required. We will review the strategies currently followed to engineer a professional lytic virus for epithelial cells into an innocuous gene delivery vehicle. Potential effects on the transducing properties of the vector that may result from the inactivation of viral activities that normally allow/regulate extrachromosomal gene expression during wild-type infection are discussed.

Adenovirus E1 Proteins↗
PubMed1997

Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses.

We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity. Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression. A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice. The immune response to the virally encoded beta-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver. Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CpG dinucleotides. However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter may be critical to maintain transgene expression from these attenuated adenovirus vectors.

Adenoviridae↗
PubMed1996

Phase I study of a recombinant adenovirus-mediated gene transfer in lung cancer patients.

BACKGROUND: Despite vigorous efforts at curbing tobacco consumption and aggressive combined-modality treatment programs, both the incidence of and the mortality from lung cancer have remained virtually unchanged in the last 10 years. More effective innovative therapies are clearly needed. The direct transfer into tumor cells of tumor suppressor genes or toxic gene products that specifically promote tumor cell death and spare nonmalignant cells is a potentially novel anticancer treatment approach that should be investigated. PURPOSE: On the basis of compelling preclinical data, we initiated a phase I study involving six patients with inoperable lung cancer and an endobronchial lesion accessible by bronchoscopy. Our purpose was to evaluate the feasibility, tolerance, and clinical, biologic, and immunologic effects of the intratumoral administration of a recombinant, replication-deficient adenovirus (rAd.RSV beta-gal), using the Rous sarcoma virus promoter to drive transcription of the Escherichia coli lacZ marker gene that encodes for the bacterial enzyme beta-galactosidase (beta-gal). METHODS: From June 1994 through April 1995, six patients (five males and one female) were enrolled in the trial. A single dose of recombinant virus suspension containing 10(7) or 10(8) plaque-forming units (PFU) was injected intratumorally into two successive cohorts of three patients. Eligible patients received concomitant chemotherapy. Patients were kept under isolation conditions from 3 days before the injection was given until virus excretion was undetectable. Biopsy specimens of the tumor and surrounding mucosa were collected on the 8th day and at 1, 2, and 3 months after injection. They were analyzed by cell culture, polymerase chain reaction (PCR), and beta-gal expression for the presence of recombinant adenovirus. So that the risk of virus recombination or complementation could be minimized, wildtype adenovirus carriers among the hospital staff (identified by PCR) were excluded from contact with patients who were potentially excreting recombinant virus. RESULTS: beta-gal was expressed in tumor biopsy specimens of three patients (one who received the 10(7) PFU dose level and two who received 10(8)). Bronchoalveolar lavage specimens collected immediately after injection were positive for recombinant adenovirus when analyzed in culture and by PCR. All biologic fluids were negative for recombinant virus as judged by PCR after day 12, with the exception of bronchoalveolar lavage specimens (positive PCR up to 90 days in two of three patients treated with 10(8) PFU). The blood samples obtained from the three patients treated with 10(8) PFU showed positive PCR results immediately after virus injection. Patients were kept in isolation for a median of 17 days. The most common toxic effects were moderate bleeding (occurring in two patients) during bronchoscopy and fever (seen in four patients). Endoscopic and clinically objective antitumor responses were seen in four patients, including one patient who showed a complete response by pathologic evaluation. The median survival for the patients was 12.5 months (range, 3-16+ months). Throughout the study, hospital staff remained negative for recombinant adenovirus infection. CONCLUSIONS: This ongoing phase I study has demonstrated that a recombinant adenovirus-mediated marker gene, such as rAd.RSV beta-gal, can be safely introduced into humans and that the gene product is expressed by lung tumor cells of the host.

Adenoviridae↗
PubMed1996

In vitro and in vivo hepatoma cell-specific expression of a gene transferred with an adenoviral vector.

Recombinant adenoviruses are widely used for the transfer of foreign genes into various mammalian cells. However, the utilization of these vectors for cancer gene therapy requires the specific and efficient expression of the transferred gene in tumor cells. To obtain targeted expression in hepatoma cells, we constructed recombinant adenoviral vectors containing transcriptional elements from either the rat alpha-fetoprotein (AFP) or the human insulin-like growth factor II (IGFII) genes driving expression of the nuclear beta-galactosidase gene (nls lacZ). In vitro infection revealed that the AFP but not the IGFII transcriptional regulatory sequence controlled nls lacZ expression specifically in hepatoma cells. The same specificity was obtained in vivo in subcutaneous human hepatic tumors generated by engraftment of Huh7 hepatoma cells in nude mice as well as in primary liver tumors developed in rats and mice. No marker gene expression was detectable after AFP-nls lacZ gene transfer to normal rat liver parenchyma despite evidence for the presence of DNA encoding the nls lacZ gene. However, in vivo experiments with primary liver tumors in rats and mice also revealed that primary hepatoma cells were poorly infected by adenoviral vectors. Peritumoral and normal tissues were infected efficiently by adenoviral vectors. We conclude that hepatoma cell-specific expression of a transgene can be achieved with AFP regulatory sequences but that adenoviral vectors may not be the preferable vector for transferring genes in vivo in primary liver tumors.

Adenoviridae↗
PubMed1996

Induction of a cytolytic T-cell response in mice with a recombinant adenovirus coding for tumor antigen P815A.

We investigated the efficacy of a recombinant adenovirus in inducing a cytolytic T-lymphocyte (CTL) response in mice against tumor antigen P815A, which is present on mouse mastocytoma P815. The recombinant adenoviral vector (Adeno.PIA) contained the sequence coding for the antigenic nonapeptide which binds to the H-2.Ld molecule to form antigen P815A. We verified that murine cells infected in vitro with Adeno. PIA were lysed by an anti-P815A CTL clone. Mice then received a single intradermal injection of Adeno. PIA, and after a few weeks their spleen cells were stimulated in vitro with tumor cells expressing antigen P815A. An anti-P815A CTL response was observed with the spleen lymphocytes of nearly all the mice, providing the lymphocytes were re-stimulated in vitro with cells expressing both P815A and co-stimulatory molecule B7.1. When the stimulatory cells did not express B7.1, a specific CTL response was observed in only 45% of the mice, and it was less intense. The Adeno. P1A viral vector was unable to raise an anti-P815A response in mice that had been previously infected with a recombinant adenovirus carrying the beta-galactosidase gene or with a defective adenovirus. We conclude that adenoviral vectors may be very useful for the priming of cytolytic T-cell responses directed against human tumor antigens. Other modes of immunization may be necessary to boost the responses induced with adenoviral vectors.

Adenoviridae↗
PubMed1996

Functional protection of dystrophic mouse (mdx) muscles after adenovirus-mediated transfer of a dystrophin minigene.

Fast skeletal muscles of mdx (X chromosome-linked muscular dystrophy) mice were injected after birth with a recombinant adenovirus containing a minidys- trophin gene, a 6.3-kbp cDNA coding for the N- and C-terminal ends of dystrophin. Adult muscles were challenged by forced lengthening during tetanic contractions. Stretch-induced mechanical and histological damages were much reduced in injected muscles, in direct proportion of the Miniber of fibers expressing minidystrophin. Damaged fibers were preferentially found among minidystrophin-negative regions. Minidystrostrophin confers an important functional and structural protection of limb muscles against high mechanical stress, even after a partial somatic gene transfer.

Adenoviridae↗