Biomedical subjects

M Perricaudet

Publications and source records attributed to M Perricaudet.

At least 91 records · Page 5Linked to original sources

PubMed1995

CFTR gene transfer corrects defective glycoconjugate secretion in human CF epithelial tracheal cells.

We demonstrate that in immortalized normal human tracheal epithelial cells (NT-1 and 56FHTE8o-) 14C-labeled glycoconjugate secretion may be regulated independently by agonists of the protein kinase A (PKA) and protein kinase C (PKC) signaling pathways. In contrast, in immortalized cystic fibrosis (CF) human tracheal epithelial cells (CFT-1 and CFT-2), regulation is defective for agonists specific for the PKA but not for the PKC pathway. To characterize the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in regulated glycoconjugate secretion, we examined the effect of adenovirus-mediated gene transfer of CFTR to CF and control cells. Forty-eight hours after infection, at a multiplicity of infection of 50 plaque-forming units per cell, high levels of CFTR mRNA were detected by reverse transcription-polymerase chain reaction, and de novo synthesis of CFTR protein was demonstrated by immunoblotting. Gene transfer to CF cells restored defective adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretion not only of chloride but also of glycoconjugates. Taken together, these results argue for a role for CFTR in cAMP-mediated glycoconjugate secretion.

Adenoviridae↗

PubMed1995

Polarity of secretion of alpha 1-antitrypsin by human respiratory epithelial cells after adenoviral transfer of a human alpha 1-antitrypsin cDNA.

alpha 1-Antitrypsin (alpha AT) deficiency, a hereditary cause of progressive emphysema, can potentially be treated by transfer of a functional human alpha 1AT gene to the respiratory epithelium. For such an approach to be successful, alpha 1AT must be provided to both the interstitium and the epithelial surface--that is, the alpha 1AT directed by the transferred gene must be secreted to both the apical and basolateral surfaces of the epithelial cells. To evaluate this concept, a recombinant, replication-deficient adenoviral vector (Ad-alpha 1AT) containing a human alpha 1AT cDNA driven by an adenovirus major late promoter was used to infect Bet-1A human respiratory epithelial cells. The infected cells expressed Ad-alpha 1AT-directed mRNA transcripts and synthesized and secreted functional human alpha 1AT as shown by [35S]methionine labeling and immunoprecipitation of a 52-kD glycosylated human alpha 1AT molecule capable of interacting with neutrophil elastase, its natural substrate. Bet-1A cells grown on microporous polycarbonate membranes formed tight junctions (resistance > 150 omega x cm2). After infection with Ad-alpha 1AT, [35S]methionine labeling and enzyme-linked immunoassay demonstrated that alpha 1AT was secreted into both the apical and basolateral compartments, with an average apical to basolateral ratio of 1.9 +/- 0.2. Thus, human respiratory epithelial cells infected with a recombinant adenoviral vector containing a human alpha 1AT cDNA secrete alpha 1AT across both the apical and basolateral cell membranes, suggesting that the respiratory epithelium could serve as a target for in vivo gene therapy of alpha 1AT deficiency.

Adenoviridae↗

PubMed1995

Low-efficiency of percutaneous adenovirus-mediated arterial gene transfer in the atherosclerotic rabbit.

Recombinant adenoviruses are the most efficient vectors with which to perform arterial gene transfer. Previous in vivo studies of adenovirus-mediated arterial transfection, however, have been performed using normal or endothelium-denuded arteries. It is unclear whether these results can be extended to atherosclerotic arteries. Accordingly, this study was designed to (a) assess the feasibility of adenovirus-mediated gene transfer to atherosclerotic lesions, and (b) compare the transfection efficiency, anatomic distribution of transfected cells, and duration of transgene expression achieved in normal versus atherosclerotic arteries. A recombinant adenovirus including a nuclear-targeted beta-galactosidase gene was percutaneously delivered to the iliac artery of normal (n = 25) and atherosclerotic (n = 25) rabbits. Transgene expression, assessed by morphometric as well as chemiluminescent analyses, was documented in all normal and atherosclerotic arteries between 3 and 14 d after gene transfer, but was undetectable at later time points. Transfected cells were identified as smooth muscle cells located in the media of normal arteries, and in the neointima and the vasa-vasora of atherosclerotic arteries. Two percent of medial cells, but only 0.2% of medial and neointimal cells expressed the transgene in normal and atherosclerotic arteries, respectively (P = 0.0001). Similarly, nuclear beta-galactosidase activity was higher in normal than in atherosclerotic arteries (3.2 vs. 0.8 mU/mg protein, P = 0.02). These findings indicate that atherosclerosis reduces the transfection efficiency which can be achieved with adenoviral vectors, and thus constitutes a potential limitation to adenovirus-based, arterial gene therapy.

Adenoviruses, Human↗

PubMed1995

The constitutive expression of the immunomodulatory gp19k protein in E1-, E3- adenoviral vectors strongly reduces the host cytotoxic T cell response against the vector.

The immune response against cells infected by gene therapy vectors may be a major hindrance for gene therapy, destroying infected cells thus limiting the length of exogene expression and quickly eliminating infected cells on repeat administration. Adenoviruses and many other pathogens have evolved strategies for escape from immune surveillance, including the gp19k gene, found in the adenovirus E3 region, known to down-regulate major histocompatibility complex class 1 expression on the cell surface, and thus reduce lysis of the infected cells by cytotoxic T cells. We have constructed an adenoviral vector expressing the genes for beta-galactosidase and gp19k both under the control of constitutive promoters, and compared the capacity of lymphocytes from DBA/2 mice previously injected with the virus or with Ad-beta gal, a virus expressing beta-galactosidase but not gp19k, to lyse target cells infected with various viruses. Lymphocytes raised against Ad-beta gal fail to lyse target cells infected with Ad-beta gal-gp19k significantly, whereas Ad-beta gal infected target cells and a beta-galactosidase expressing cell line are strongly lysed. The administration of Ad-beta gal-gp19k fails to stimulate the proliferation of anti-vector lymphocytes, and thus these lymphocytes show poor cytotoxic activity against Ad-beta gal or Ad-beta gal-gp19k infected cells.

3T3 Cells↗

PubMed1995

Organoids direct systemic expression of erythropoietin in mice.

Organoids are adenoviral vector transduced cells embedded ex vivo in a collagen-polytetrafluoroethylene lattice that is saturated with angiogenic factors. Organoids provide an alternative method of cell mediated gene transfer following implantation in the donor/recipient. The feasibility of adenovirally mediated delivery via organoids using the erythropoietin (Epo) cDNA was tested. Fibroblasts were transduced by two recombinant adenoviral vectors encoding the Macaca cynomolgus Epo cDNA, driven by a viral (RSV LTR) or a murine housekeeping gene promoter (PGK-1). A functional in vivo assay was used to monitor Epo production via the rise in hematocrit(s) (hct). The hct remained elevated for as long as 6 weeks after implantation. Subcutaneous implants gave consistently higher hct than intraperitoneal implants, while organoids made with a greater number of cells, or an equal number of cells transduced at higher multiplicities of infection (MOI) also produced a larger increase in hct. AdPGKEpo-organoids produced a greater increase in hct than AdRSVEpo-organoids under comparable conditions, but the duration of expression was similar. A 10- to 50-fold lower input of AdRSVEpo using organoids versus direct intravenous injections resulted in an equal to, or greater than hct response in mice. Explanted organoids caused a rapid decrease in the hct of mice. Organoid supernatant had little or no detectable free viral particles making this method safe from unwanted recombinant adenovirus dissemination.

Animals↗

PubMed1995

[Adenovirus, a gene therapy vector: application to lung diseases].

Due to their quiescent nature and spatial complexity, many target tissues for gene therapy will require novel strategies. An alternative to ex vivo gene transfer, providing many technical advantages and possibly allowing sufficient transfer of the therapeutic gene, is direct in vivo delivery of the vehicle. For a favorable outcome, this procedure is dependent on a high-titer vector, fully competent before post-mitotic cells. In view of the restrictions inherent in the use of retroviruses, we have investigated the potentials of adenovirus. Adenoviruses have as primary targets of infection the differentiated epithelial cell. The large DNA genome of the virus hints to a large cloning capacity. Furthermore, wildtype adenovirus and the precedent of administration to humans are suggestive of adenovirus-based gene therapy for diseases involving a variety of quiescent tissues. The use of a replication-defective adenovirus carrying a gene encoding a nuclearly-targeted beta-galactosidase demonstrated that replication-incompetent adenoviruses offer an efficient means to transfer a gene for extended periods of time to the liver, muscle, lung, and brain. Because of adenovirus' natural tropism for the lung epithelium, we have proposed that this virus be used as a CFTR gene delivery vector for the treatment of cystic fibrosis and lung cancer.

Adenoviruses, Human↗

PubMed1995

Complete recovery of mice from a pre-established tumor by direct intratumoral delivery of an adenovirus vector harboring the murine IL-2 gene.

Direct introduction of exogenous genes into pre-existent tumors could provide an effective therapeutic approach for treatment of localized tumors. In this report we show that direct intratumoral delivery in animals of a replication-deficient adenovirus vector harboring the murine interleukin (IL)-2 gene (AD-mIL2) causes complete disappearance of P815 murine mastocytoma tumors in up to 75% of cases. Histological analysis of treated tumors revealed the presence of several zones of necrosis and the infiltration of macrophages and T cells. Moreover, the successfully treated animals develop a long lasting state of immunity during which further challenges with the tumor cells are rejected. To our knowledge this is the first successful in vivo treatment of an established tumor using adenoviral gene therapy methods.

Adenoviruses, Human↗

PubMed1994

Direct intracerebral gene transfer of an adenoviral vector expressing tyrosine hydroxylase in a rat model of Parkinson's disease.

Direct intracerebral gene transfer to neural cells has been demonstrated with recombinant adenovirus encoding beta-galactosidase. To explore the potential of recombinant adenovirus for the therapy of neurological disease we constructed a recombinant adenovirus encoding tyrosine hydroxylase and optimized intracerebral injection to express the gene in the striatum of unilaterally denervated rats. These animals have dopamine depletion in their lesioned striatum, causing a rotation asymmetry induced by apomorphine. One and two weeks after intracerebral injection this sensorimotor asymmetry was decreased by the adenovirus encoding tyrosine hydroxylase and not by a control adenovirus encoding beta-galactosidase. Histological analysis showed that tyrosine hydroxylase was preferentially expressed in astrocytes.

Adenoviridae↗

PubMed1994

Adenoviral vectors as functional retrograde neuronal tracers.

Adenoviruses have been recently recognized as a highly efficient system for gene delivery to various tissues. The ability of replication-defective recombinant adenovirus to transfer the lacZ reporter gene encoding beta-galactosidase to nerve cells in various brain structures has been demonstrated. Here, on the continuation of these studies, we present evidence that the adenovirus can be transported in a retrograde manner to nerve cell bodies from axonal terminals. This method may be of great value for infecting selected subsets of specific neurons for either anatomo-functional studies or even therapeutic purposes.

Animals↗

PubMed1994

The use of adenovirus vectors for intracerebral grafting of transfected nervous cells.

Grafting genetically-modified cells into the brain is a promising approach to address fundamental and clinical issues in neurobiology. Despite recent substantial progress, most of the methods used for introducing DNA sequences into donor cells result in weak efficacy or transient gene expression after transplantation. We tested whether the use of adenovirus as the vector for foreign genes avoided these problems. A replication-defective adenovirus vector carrying a reporter gene encoding for beta-galactosidase was used to transfect primary astrocytes. After grafting into various brain structures, transfected cells exhibited robust survival and expressed the transgene for at least five months. These results demonstrate the advantage of adenovirus-mediated gene transfer for prolonged transgene expression in grafted primary cells.

Adenoviridae↗

PubMed1994

The Epstein-Barr virus determined nuclear antigens EBNA-3A, -3B, and -3C repress EBNA-2-mediated transactivation of the viral terminal protein 1 gene promoter.

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) has been shown to transactivate both cellular and viral gene promoters including the promoter for the viral terminal protein 1 gene (TP-1). We investigated whether three other EBV nuclear antigens EBNA-3A, -3B, and -3C (which themselves share a degree of primary sequence homology) could also play a role in TP-1 gene regulation. The TP-1 promoter sequence was linked to the chloramphenicol acetyltransferase (CAT) gene and used in cotransfection experiments in an EBV negative cell line with various combinations of vectors expressing individual EBNA-3s. In the absence of other EBV proteins, the EBNA-3s did not stimulate TP-1 promoter activity. In the presence of EBNA-2, the EBNA-3s were shown to be capable of reducing the level of TP-1 promoter-driven CAT activity. The EBNA-3s had no effect on a panel of heterologous promoters, indicating that EBNA-2 and/or transcription elements specific to the TP-1 promoter are essential for the observed activity of the EBNA-3s. The functional antagonism between the EBNA-2 and EBNA-3 proteins may be important in the overall viral strategy.

Antigens, Viral↗

PubMed1994

Adenoviral-mediated gene transfer to renal tubular cells in vivo.

The efficient introduction of genetic material into quiescent renal cells is potentially important in the study of renal physiopathology and for gene therapy of kidney related disorders. A replication-deficient adenoviral vector that contained a reporter gene encoding the nuclear beta-galactosidase was either selectively perfused into the renal artery or infused through a retrograde catheter into the pyelic cavity of the left kidney of adult rats. Highly efficient gene transfer was achieved by either route of administration, and nuclear beta-galactosidase activity was detected for two to four weeks following a progressive decline of expression. Genetically-modified cells were identified as proximal tubular cells when the adenoviral vector was selectively perfused via the renal artery, while tubular cells from the papilla and medulla were selectively transduced by retrograde infusion of the viral vector. No obvious cytopathic effect was observed. We conclude that: (i) efficient gene transfer in renal tubular cells can be achieved by adenoviral vectors; (ii) the targeted cell population can be chosen through the route of administration.

Adenoviruses, Human↗

PubMed1994

Lung gene therapy: in vivo adenovirus-mediated gene transfer to rhesus monkey airway epithelium.

Somatic gene therapy of lung disorders such as cystic fibrosis (CF) aims at introducing the therapeutic gene into respiratory epithelium. We have tested the ability of recombinant human adenovirus to infect rhesus monkey airway epithelium in vivo. Application of adenovirus harboring the lacZ marker gene to the airway surface resulted in large patches of lacZ-positive cells in the trachea, bronchi, and bronchioles, 6 days after virus exposure, indicating a successful transfer of the lacZ gene to respiratory epithelium. Microscopic analysis showed that basal, mucous goblet, and ciliated cells were lacZ positive. In addition, gene transfer to the submucosal glands was observed. Pathological examination of the organs revealed no virus-mediated toxic effects to the lungs and other organs. Using polymerase chain reaction (PCR) analysis we found no spread of the virus to blood or any organ tested. These results indicate the potential use and safety of adenoviruses as a tool in human gene therapy procedures aimed at pulmonary diseases.

Adenoviruses, Human↗

PubMed1994

Erythropoietin gene transfer and expression in adult normal mice: use of an adenovirus vector.

A hormonal model of erythropoietin (Epo) delivery by use of an adenovirus vector was investigated. We constructed a replication-defective adenovirus carrying the monkey (cynomolgus) Epo cDNA under control of the Rous sarcoma virus long terminal repeat promoter. Fifty 8-week-old mice were injected with escalating doses of the recombinant virus from 10(6) to 10(10) plaque-forming units (pfu). Different modes of administration were studied. Intravenous (i.v.) injection was the most effective mode of administration and exhibited a dose-dependent response. After a single i.v. injection with high doses (5 x 10(9) and 10(10) pfu), a dramatic increase in hematocrit (Hct) and long-term Epo expression (6 months at this time) were observed. Intravenous administration with lower doses and intramuscular (i.m.) administration were inefficient or had a very transient effect. A localized muscle attrition prior to i.m. administration of 10(10) pfu enhanced Hct response. This initial study opens the way for high level and durable Epo therapy by gene transfer. Moreover, this recombinant virus provides a convenient means to study the efficacy, duration, and safety aspects of hormonal delivery by an adenoviral vector.

Adenoviridae↗

PubMed1994

Gene transfer to human fetal pulmonary tissue developed in immunodeficient SCID mice.

Human fetal lung rudiments (8-12 weeks of development) undergo considerable growth upon microsurgical ectopic implantation in the xenograft-tolerant SCID mouse, and differentiate into a lung-like tissue that includes: (i) bronchial structures lined with pseudostratified, secretory, ciliated epithelium surrounded by smooth muscle and cartilage rings, (ii) submucosal glands, and (iii) alveolar sacs. Normal expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was detected by immunostaining in those grafts, and similar differentiation was observed from either normal or cystic fibrosis (CF) fetal lung rudiments. Upon microinjection into human CF or normal lung grafts in SCID mice, beta-galactosidase-adenovirus gene constructs were efficiently transduced into epithelial and glandular cells. Such an in vivo replica of the human respiratory tissue may be a useful experimental model to study normal and pathologic lung development, and to assay candidate therapeutic gene constructs preclinically.

Adenoviridae↗

PubMed1994

Arterial gene transfer to rabbit endothelial and smooth muscle cells using percutaneous delivery of an adenoviral vector.

BACKGROUND: Previous investigations in live animals convincingly established that arterial gene transfer, while feasible, was compromised by a low transfection efficiency. More recent studies have shown that transfection efficiency may be substantially augmented by the use of recombinant adenoviral vectors. Most in vivo transfections reported to date, however, have used direct (operative) administration of the adenoviral vector. Clinical applications of arterial gene transfer (such as prevention of restenosis), however, would require local percutaneous delivery of the transgene. The present study was designed to extend in vivo intraoperative findings to percutaneous delivery system and to assess whether gene transfer remains site specific. METHODS AND RESULTS: A recombinant, replication-defective adenovirus modified to include an expression cassette for nucleus-targeted beta-galactosidase was introduced into rabbit iliac arteries in vivo using either a double-balloon catheter (DBC, n = 27) or a hydrogel-coated balloon catheter (HBC, n = 27). Contralateral arteries-normal, endothelium-denuded, or sham-transfected with a control adenoviral vector-served as controls. beta-Galactosidase expression was assessed by X-Gal staining. Cell-transduction efficiency was measured by morphometric analysis. Polymerase chain reaction (PCR) and histochemistry were used to detect the presence and/or expression of viral DNA in remote organs. Transgene expression was detected in all cases (46 of 46) between 3 and 14 days after transfection but was in no case detectable 28 days after transfection. In the DBC group, transgene expression was limited to endothelial cells when the endothelium was left intact and to rare medial cells (< 2.2%) when it had been removed. In contrast, HBC delivery resulted in transduction of up to 9.6% of medial smooth muscle cells (P = .0001). Optimized PCR and histochemistry failed to detect evidence of extra-arterial transfection except in a small number of cells (between 1 in 3 x 10(2) and 1 in 3 x 10(5) cells) in the livers of 2 animals in the DBC group. CONCLUSIONS: (1) Efficient, adenovirus-mediated, arterial gene transfer to endothelial and/or smooth muscle cells is feasible by percutaneous, clinically applicable techniques. (2) Consistent transfection of medial smooth muscle cells may be achieved when the endothelial layer is abraded. (3) Medial transfection is more efficient when an HBC, rather than a DBC, is used. (4) Percutaneous delivery of the adenoviral vector via HBC results in site-specific arterial gene transfer. Very-low-level extra-arterial transfection may occur, however, when the DBC is used.

Adenoviridae↗

PubMed1994

In vivo adenovirus-mediated gene transfer into ocular tissues.

Replication-deficient adenoviruses have been successfully used to transfer foreign DNA into a variety of cells including post-mitotic cells, in vivo. In the eyes, most of the cells are quiescent or slowly dividing. They constitute the obligatory targets of gene transfer for a number of ocular diseases that have been elucidated at the molecular level and are potential targets for gene therapy. We have therefore analysed the ability of an adenovirus vector to transfer in vivo the Escherichia coli lacZ gene into ocular cells of mice. Injection of up to 3 x 10(7) p.f.u. into the vitreous body, the anterior chamber or the peribulbar space, did not result in any detectable cytopathic effect and was associated with endocytosis of viral particles in corneal endothelial, photoreceptor, bipolar, ganglionic and oculomotor muscle cells, depending on the administration route. At the viral titer used (3 x 10(7) p.f.u.), the expression was detected for at least 50 days. These results open new prospects for the treatment of some retinal hereditary disorders and acquired corneal or retinal alterations due to inflammatory disease.

Adenoviridae↗

PubMed1994

In vivo adenovirus-mediated transfer of human CFTR cDNA to rhesus monkey airway epithelium: efficacy, toxicity and safety.

We have administered replication defective recombinant adenovirus harboring the human CFTR cDNA (Ad.CFTR) to lungs of Rhesus monkeys and assessed toxicity, efficiency of gene transfer and containment of recombinant adenovirus in the lungs. Gene transfer efficiencies, as measured by PCR analysis, were dose-dependent. Administration of low dose Ad.CFTR [1.5 x 10(7) plaque forming units (p.f.u.)] was not accompanied by any clinical chemical or histopathological changes. Mild to moderate multifocal perivascular and peribronchial lymphocytic infiltrates were found upon histopathological analysis only after administration of a high dose of recombinant adenovirus, although not accompanied by changes in clinical chemical parameters. Long-term expression (up to 128 days) was found after administration of recombinant adenovirus. Re-challenging of the monkeys treated with high-dose recombinant adenovirus resulted again in gene transfer at all levels of lungs and airways, without being accompanied by additional histopathological changes. Circulating anti-adenovirus antibodies were elicited. Animals treated with high-dose adenovirus secreted virus in pharynx and faeces for maximally 2 and 4 days after administration, respectively. These results show that recombinant adenovirus can be used for efficient delivery of genes into primate airway epithelium without signs of severe toxicity.

Adenoviridae↗