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M Perricaudet

Publications and source records attributed to M Perricaudet.

At least 73 records · Page 4Linked to original sources
PubMed1996

Keratinocytes as a target for gene therapy. Sustained production of erythropoietin in mice by human keratinocytes transduced with an adenoassociated virus vector.

BACKGROUND AND DESIGN: Keratinocytes are ideal targets for somatic gene therapy. Among the viral gene transfer systems, adenoassociated virus vectors have recently gained attention. We studied the feasibility of using adenoassociated virus-transduced human keratinocytes to provide a long-term, high-level production of a therapeutic factor after implantation in mice. RESULTS: Transduction of HeLa cells by an adenoassociated virus vector was ascertained by transfer of the beta-galactosidase reporter gene, which was visualized by the blue staining of infected cells after fixation and coloring by X-Gal (the substrate of the reaction for beta-galactosidase activity). In a second step, 2 HeLa cell lines transduced with an AAV harboring the erythropoietin complementary DNA and producing high amounts of erythropoietin in vitro were isolated. After implantation in nude mice, a high-level and long-term increase in hematocrit (for the 1-month duration of the study) was found, which was correlated to the size of the induced tumor. CONCLUSIONS: Adenoassociated virus-transduced HeLa keratinocytes provide high-level, stable, and long-term production of a therapeutic protein in mice. These results must now be extended to human primary keratinocytes.

Animals↗
PubMed1996

Strategies for cancer gene therapy using adenoviral vectors.

Modification of tumor cells using gene transfer either to enhance host immunity or to act directly on tumor cells is being intensively studied in animal models. Remarkable results have yielded to approved clinical protocols in the treatment of cancer patients using this approach. Several methods of gene delivery have been developed. This article is particularly devoted to the interest of the use of adenoviral vectors in the different strategies of cancer gene therapy.

Adenoviridae↗
PubMed1996

Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit.

Transgene expression after the administration of recombinant adenovirus with E1 deleted is constantly transient. It is admitted that E1A-substituting activities of cellular or viral origin allow viral antigen synthesis and trigger cytotoxic lymphocyte-mediated clearance of the recipient cells. Our approach to solving this problem relies on the additional deletion of the E4 region from the vector backbone as this region upregulates viral gene expression at both transcriptional and posttranscriptional levels. As a prerequisite to the construction of E1 E4 doubly defective adenoviruses, we investigated the possibility of transcomplementing both functions within a single cell. In particular, the distal ORF6+ORF7 segment from the E4 locus of adenovirus type 5 was cloned under the control of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Following transfection into 293 cells, clone IGRP2 was retained and characterized as it can rescue the growth defect of all E1+ E4- adenoviral deletants tested. DNA and RNA analysis experiments verified that the mouse mammary tumor virus promoter drives the expression of the ORF6+ORF7 unit and permits its bona fide alternative splicing, generating ORF6/7 mRNA in addition to the ORF6-expressing primary transcript. Importantly, IGRP2 cells sustain cell confluence for a period longer than that of 293 parental cells and allow the plaque purification of E1- or E4- defective viruses. The dual expression of E1 and E4 regulatory genes within IGRP2 cells is demonstrated by the construction, plaque purification, and helper-free propagation of recombinant lacZ-encoding doubly defective adenoviruses harboring different E4 deletions. In addition, the emergence, if any, of replicative particles during viral propagation in this novel packaging cell line will be drastically impaired as only a limited segment of E4 has been integrated.

Adenovirus E1 Proteins↗
PubMed1996

Epstein-Barr virus EBNA3A and EBNA3C proteins both repress RBP-J kappa-EBNA2-activated transcription by inhibiting the binding of RBP-J kappa to DNA.

Following infection by Epstein-Barr virus (EBV), the production of viral nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C and the viral membrane protein LMP1 is essential for the permanent proliferation of primary B lymphocytes to occur. Among these, the transcription factor EBNA2 is central to the immortalizing process, since it activates not only the transcription of all the EBNA proteins and LMP1, TP1, and TP2 but also certain cellular genes. EBNA2 is targeted to its DNA-responsive elements through direct interaction with the DNA-binding cellular repressor RBP-J kappa. In a transient-expression assay, the EBNA2-activated transcription was found to be downregulated by EBNA3A, EBNA3B, and EBNA3C. However, since it has been reported that EBNA3C, but not EBNA3A, directly contacts RBP-J kappa in vitro, these proteins appear to repress through different mechanisms. Here, we report for the first time that EBNA3A and EBNA3C both stably interact with RBP-J kappa and most probably repress EBNA2-activated transcription by destabilizing the binding of RBP-J kappa to DNA.

Antigens, Viral↗
PubMed1996

LacZ gene transfer into tumor cells abrogates tumorigenicity and protects mice against the development of further tumors.

Numerous studies have shown that the expression of immuno-stimulatory genes in tumor cells can result in the development of antitumoral immunity resulting in the rejection of the tumor cells. We show here that the simple integration and expression of the lacZ gene in the highly tumorigenic murine mastocytoma cell line P815 strongly reduces the cell's tumorigenicity. All the animals having rejected P815-lacZ challenges develop a long-lasting immunity against unmodified P815 cells with all of the animals rejecting further challenges with tumorigenic doses of P815 cells. However, this protective immunity conferred by P815-lacZ, directed against both the nuclearly expressed lacZ and surface tumor antigens, is not sufficient to act as curative immunity. In this immunogenic tumor model the expression of the lacZ antigen is more efficient than the irradiation of the cells to induce a strong immune response and an antitumoral state of vaccination in syngeneic animals.

Animals↗
PubMed1996

Gene therapy of rat C6 glioma using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene: long-term follow-up by magnetic resonance imaging.

The herpes simplex virus thymidine kinase gene was transferred into C6 glioma cells by infection with a recombinant adenovirus. In vitro, a 10 microM ganciclovir concentration was able to kill 100% of the infected cells. For in vivo experiments, brain tumors were established by stereotactic injection of C6 glioma cells in the caudate nucleus of rats. Five days later, the recombinant adenovirus was inoculated into the tumors and the animals were treated by intraperitoneal injections of ganciclovir for 14 days. At the end of ganciclovir therapy, histological examination revealed a 28-fold decrease in tumor volumes in the treated animals, as compared with control animals. In long-term studies, the mean survival time of the treated animals were four-fold longer than that of control ones. Magnetic resonance imaging demonstrated an apparent complete tumor regression in 62% of the animals. However, late tumor recurrence was observed in the treated animals. Repeated inoculation of C6 glioma cells in the contralateral hemisphere of long-term surviving animals resulted in either tumor rejection or slowly growing tumors. These findings demonstrate the potential efficacy of adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene and ganciclovir administration in the treatment of rat gliomas.

Adenoviridae↗
PubMed1996

Restoration of hexosaminidase A activity in human Tay-Sachs fibroblasts via adenoviral vector-mediated gene transfer.

Tay-Sachs disease (TSD) is a lysosomal storage disease due to hexosaminidase A deficiency caused by mutations in the gene for alpha-chain (Hex alpha). A human Hex alpha cDNA was subcloned into the adenoviral plasmid pAdRSV. Hex alpha. Replication-deficient adenovirus was generated by homologous recombination in 293 cells. Human fibroblasts from a patient suffering from TSD were infected with the recombinant adenovirus. TSD fibroblasts expressing the recombinant alpha-chain had an enzyme activity on the natural substrate ranging from 40 to 84% of the normal. The corrected cells secreted up to 25 times more Hex alpha than control fibroblasts. The Hex alpha encoded by the adenovirus was shown to be correctly transported into the lysosomes and to normalize the impaired degradation of GM2 ganglioside in TSD fibroblasts.

Adenoviridae↗
PubMed1996

[Development of adenoviral vectors in gene therapy: application to gene transfer in muscles].

This review focuses on the general properties of adenovirus vectors and their application to muscle gene transfer. In the prospect of a treatment for Duchenne muscular dystrophy, in vivo expression of the dystrophin gene in animal models remains the main concern. After the presentation of the two partners: adenovirus and muscle, we summarize our results of gene transfer in mice. We have evaluated efficiency of systemic and intramuscular injections, the impact of age at injection, the duration of expression in adult normal and genetically-immunosuppressed SCID mice, using a recombinant adenovirus expressing a reporter gene. After adenovirus-mediated transfer of a mini-model, we have shown an efficient and stable expression of the transgene, a long-term correction of the degeneration process and a functional protection of the treated muscle. The discussion focuses on the problems and the perspectives for gene therapy: safety problems, improvement of safety and vector capacity, host immune response, delivery to muscle and muscle-targeted expression.

Adenoviridae↗
PubMed1996

Heart muscle-specific gene expression using replication defective recombinant adenovirus.

Adenoviruses are a promising vector system for future gene therapy of heart muscle diseases. The promiscuous tissue tropism of adenoviruses, however, may lead to the undesirable expression of putative therapeutic genes in nontarget cells and hence to considerable safety limitations for this vector system. To restrict gene expression to cardiomyocytes we constructed an adenoviral vector (Ad-mlcLuc) in which the luciferase gene is under the control of the ventricle-specific myosin light chain-2 (mlc-2v) promoter. For controls, we constructed a recombinant adenovirus without promoter (Ad-Luc) and one with the Rous sarcoma virus (RSV) promoter (Ad-rsvLuc). Our data demonstrate that the newly established viral vector Ad-mlcLuc was specifically active in rat neonatal cardiomyocytes in vitro but not in three established cell lines. Injections of the recombinant adenoviruses into the cardiac cavity of neonatal rats resulted in myocardial specific gene expression of Ad-mlcLuc in vivo, despite the fact that viral DNA was detected by PCR at different levels in all tissues investigated. In vitro and in vivo, Ad-mlcLuc was exclusively active in cardiac muscle cells, reaching 8-9% of the RSV-induced luciferase activity. Direct injection of Ad-mlcLuc into thigh muscle gave only background luciferase activity (0.05% of Ad-rsvLuc). Therefore, in the adenoviral system, the mlc-2v promoter allows heart-specific expression of a foreign gene thus providing a promising tool for gene transfer targeted to the myocardium.

Adenoviruses, Human↗
PubMed1996

Adenovirus vector-transduced hepatocytes implanted via a preformed collagen/PTFE support persist for at least 4 weeks in vivo.

Liver-directed gene therapy has the potential to provide an effective adjutant to conventional treatments for liver diseases. In vivo gene transfer is promising but still effectively out of reach with conventional gene delivery techniques. Ex vivo gene therapy using hepatocyte transplantation, however, has been encouraging. We describe and approach toward treatment of liver-related diseases by combining several of the advantageous properties of current liver-directed therapies. Primary hepatocytes were isolated, transduced with an adenovirus vector encoding a reporter gene, embedded in a collagen/polytetrafluoroethylene (PTFE) lattice, and implanted in mice. Recovered 'hepatocyte-organoids' were assayed for the presence and viability of the implanted hepatocytes, duration of transgene expression and presence of the adenovirus vector. In an initial attempt, we demonstrate that genetically modified hepatocytes can survive and express a transgene for at least 4 weeks in vivo when embedded in a collagen/PTFE support and implanted in the intraperitoneal cavity. This approach takes advantage of hepatocyte-specific functions in order to treat diseases where a fraction of the normal enzymatic activity is sufficient to alleviate a disease phenotype.

Adenoviridae↗
PubMed1996

Dynamics of gene transfer to retinal pigment epithelium.

PURPOSE: To examine the nature and dynamics of gene transfer to human retinal pigment epithelium (RPE) using an adenoviral vector and adjuvants that may enhance the uptake of recombinant adenoviruses. METHODS: Human RPE cultures (HRPE7) were transfected in vitro with varying concentrations (4, 20, 40, 120, and 200 pfu/microliter) and for varying periods (1, 2, 4, 16, 24, 48, and 72 hours) with a replication-deficient adenovirus (Ad.RSV. beta gal) containing the bacterial beta-galactosidase transgene (beta gal). The expression of beta gal was monitored by counting after X gal staining. The transgene expression profiles were compared to those of human F2000 fibroblasts under the same conditions. The adjuvant effect of sodium hyaluronate (HA) on the expression of beta gal was tested in F2000 and early and late passage human RPE cells for differing concentrations of HA, viral titers, and incubation times. Immunofluorescent cytochemistry was carried on HRPE7 and F2000 cells for the HA receptors, homing receptor CD44 (CD44), intercellular adhesion molecule 1 (ICAM-1), and the receptor for hyaluronan mediated motility (RHAMM). RESULTS: The number of HRPE7 and F2000 cells expressing the adenoviral transgene increased consistently with increasing incubation time and viral titer. There was a higher uptake of Ad.RSV. beta gal in HRPE7 cells compared to the F2000 fibroblasts under the same conditions. There was an increase of 28.1% and 41.4% in the number of RPE7 cells expressing adenoviral transgene and 16.2% and 15.8% F2000 fibroblast cells expressing the adenoviral transgene in the presence of 0.001% and 0.005% HA, respectively. Significant adjuvant effects on transgene expression also were shown in HRPE51 cells. It appears that the effects of increasing viral titer, length of incubation, and the presence of HA on transgene expression are at least additive. The appearance of CD44 and ICAM receptors on RPE7 and F2000 cells and RHAMM receptors on F2000 cells was similar. The RHAMM receptors in HRPE7 cells, however, were shown preferentially over the nucleus. CONCLUSIONS: On the basis of these results, the authors propose that adenovirus transgene expression increases with increasing incubation time and viral titer in cell culture. The rate of increase of expression differs between human RPE cells and the F2000 fibroblast cells, which may offer a targeting opportunity. The authors propose that the use of HA can offer both an adjuvant effect and a targeting advantage in terms of transferring adenoviral transgenes to human RPE in culture.

Adenoviruses, Human↗
PubMed1995

Recombinant adenoviruses as vaccines.

Over the past 15 years, advances in molecular biology have permitted in vitro and in vivo gene transfer into mammalian cells. Genetically engineered microorganisms are highly promising developments for gene therapy and the future of vaccines. Such vectors constitute genuine tools for high level expression of heterologous genes for both therapeutic and induced immunity applications. Several vector systems have emerged with different relative advantages and limits depending on the proposed application. Adenovirus (Ad) has gained our interest, and in this review we focus on its applicability as a vaccine vector. We describe its potentials, as well as some of the foreseen obstacles related essentially to its use in preventive medicine.

Adenoviridae↗
PubMed1995

Adenovirus mediated gene transfer in organotypic brain slices.

A replication-defective adenovirus vector carrying the reporter gene encoding beta-galactosidase was used to transfect organotypic slices maintained in culture for up to 1 month. Three different delivery systems were used to inoculate the viral solution, either into the culture medium, or directly onto the surface of the slices or by microinjection into the tissue. Using the two first paradigms beta-galactosidase expressing cells were mostly of glial phenotype and distributed throughout the slices without any specific regional pattern. In contrast, microinjection of the adenovirus resulted in a large number of both infected neurones and glia, concentrated at the site of injection. This method thus appears to be able to circumvent some of the constraints and limitations associated with in vivo gene transfer.

Adenoviridae↗
PubMed1995

Transplantation to the rat brain of human neural progenitors that were genetically modified using adenoviruses.

Transplantations for neurological disorders are limited by the supply of human fetal tissue. To generate larger numbers of cells of appropriate phenotype, we investigated whether human neural progenitors expanded in vitro could be modified with recombinant adenoviruses. Strong expression of beta-galactosidase was obtained in vitro. Two or three weeks after transplantation of engineered cells to the rat brain, we observed a small percentage of surviving neuroblasts strongly expressing beta-galactosidase in four out of 13 rats. Thus human precursor cells that have been genetically modified using adenoviruses are a promising tool for ex vivo gene therapy of neurodegenerative diseases.

Adenoviridae↗
PubMed1995

Adenovirus and adeno-associated virus mediated gene transfer.

In this review we describe current strategies for adenoviral mediated gene transfer (AMGT) and adeno-associated viral mediated gene transfer (AAVMGT). We consider the structure and molecular biology of adenoviruses and adeno-associated viruses and detail the current advantages and disadvantages of AMGT and AAVMGT. Potential solutions to some of the specific drawbacks to AMGT, including the development of new vectors, addition of gp19k, organoides, and the use of non-human adenoviral vectors, are discussed.

Adenoviridae↗
PubMed1995

Integration of viral sequences into the c-myc gene in two mammary adenocarcinomas induced by polyomavirus in athymic nude mice.

We report the analysis of polyomavirus (Py) DNA integration into chromosomal DNA of two Py-induced mammary adenocarcinomas of athymic nude mice. Prior observations had established that these tumors had high levels of episomal Py DNA, making analysis of integration sites difficult. Propagation of tumor cells in culture allows the isolation of lines which have lost episomal Py DNA but are still tumorigenic and thus can be used for in situ and Southern analysis of Py sequences. The data reported here support the conclusion that Py DNA integrated into and next to the c-myc gene, adding further importance to this tumor system which, in its modifications of c-myc expression, appears to be similar to some human mammary cancers. In situ hybridization experiments on metaphase chromosomes of tumor cells showed that (i) in both cases, there was a single integration site at the same position on the same chromosome in all cells of a given tumor, and (ii) integration sites were different in the two tumors; in one, it was located on chromosome 15, near the c-myc proto-oncogene, and in the other, it was situated in the distal part of chromosome 1. We have demonstrated a probable rearrangement between chromosome 1 and chromosome 15, in the region of Py insertion, thus suggesting that a specific site on chromosome 15 is involved in tumorigenesis. The discovery that Py DNA was integrated at specific sites in host chromosomes raised the questions of whether such integrations were correlated with the activation of specific oncogenes. The rearrangements of the c-myc proto-oncogene observed on Southern blot analysis for both tumors, along with similar integration patterns of Py sequences, the overexpression of the c-myc gene, and the synthesis of abnormal oversized hybrid transcripts between c-myc and Py genes, favor this hypothesis. Finally, the analysis of episomal Py DNA in various tumors shows viral populations presenting a specific deletion in a part of the Py late region. This deleted region in the episomal virus genome was systematically found integrated in chromosomal DNA, thus arguing for the importance of Py integration in the induction of mammary tumor.

Adenocarcinoma↗