Search PubMed⌕ Search

Biomedical subjects

C Coutelle

Publications and source records attributed to C Coutelle.

At least 73 records · Page 4Linked to original sources
PubMed1997

Isolation of DNA from the centromere of human chromosome 7 by microdissection.

Centromeres remain the least characterized regions of human chromosomes because they have a very high content of repetitive DNA. Here, we describe a microdissection library from the centromeric region of human chromosome 7 and its use for generating sequence tagged sites (STSs). The library contains about 1500 clones with an average insert size of 150 bp and only about 15% of the clones harbour repetitive human DNA. Seven clones hybridizing to alphoid DNA were found to correspond to a fragment of the D7Z2 alphoid array on chromosome 7, thus confirming the origin of the library. A number of clones not containing known repetitive DNA were used to generate STSs that identified yeast artificial chromosomes (YACs) and in turn allowed the STSs to be placed on the physical map. One STS is located between the two Genethon genetic markers closest to the centromere on the q side. Another STS was located 3-4cM away in 7q11.2, while a third identified YACs containing both low-copy and alphoid sequences that are not yet mapped but are clearly centromeric. The library therefore comprises a collection of sequences from the centromeric region of chromosome 7 that can be used to generate STSs and to map the entire centromeric region.

Amniotic Fluid↗
PubMed1997

RT-PCR method specific for the detection of transgenic CFTR mRNA in the presence of transgene plasmid DNA and endogenous CFTR mRNA.

A major problem with experiments involving the correction of cystic fibrosis by gene transfer has been reliably detecting the transgenic message and distinguishing this from an endogenous message and from vector DNA. We have exploited the SV40 small T antigen intron present in an expression vector containing the CFTR cDNA (pCFAS) using a primer directed to the spliced sequence to allow specific and precise detection of the transgeneic message.

Antigens, Polyomavirus Transforming↗
PubMed1997

Foetal gene delivery in mice by intra-amniotic administration of retroviral producer cells and adenovirus.

With the aim of developing foetal gene therapy for cystic fibrosis, we have investigated the possibility of gene targeting to the mouse foetus with two different viral vector systems and at different times of gestation. We report here that recombinant retrovirus producing cells administered into the intra-amniotic cavity of mid- to late-gestation mouse MF1 foetuses survive in the amniotic fluid and are able to engraft to a certain extent in foetal tissues. By production of infectious virus they mediate transduction and beta-galactosidase transgene expression in neighbouring foetal tissues 24 to 72 h following injection. Retrovirus producer cells could, therefore, become a means to overcome the limitations of low retroviral titre, for in vivo foetal gene transfer. To investigate the developmental stage at which transduction of the airways and enteral systems can be obtained we also administered a highly infective first generation adenoviral vector (AdRSV beta gal) into the amniotic cavity of foetal mice between 13 to 16 days post coitus, beta-galactosidase activity was detected between 24 to 120 h after injection. The highest levels of transgene expression were generally observed between 48 to 72 h following injection of the adenoviral vector. We demonstrate that infection of the pulmonary airways is dependent on the developmental stage of the foetus and can be achieved on the 15th day of gestation.

Amnion↗
PubMed1997

Analysis of ribosomal and alphoid repetitive DNA by fiber-FISH.

We have used fluorescence in situ hybridization to extended DNA fibers (fiber-FISH) to analyse the alpha-satellite and rDNA arrays on a human chromosome 22. The rDNA could be seen as a string of signals spanning about 430 kb. The bulk of the alphoid DNA was found in a single large array of about 2.6 Mb.

Cell Line↗
PubMed1997

Recombinant HMG1 protein produced in Pichia pastoris: a nonviral gene delivery agent.

This paper describes the production of a recombinant protein from the expression system based on the methylotrophic yeast Pichia pastoris. Efficient production of rat high-mobility-group 1 (HMG1) protein was obtained using the system. Two forms of HMG1 were secreted into the culture medium: a 24.5-kDa species corresponding to the native HMG1 and a 32-kDa glycosylated derivative. Non-glycosylated recombinant HMG1 was purified easily and shown to possess the same DNA-binding properties as HMG1 purified from calf thymus. Plasmid DNA complexed to the recombinant HMG1 is taken up by a variety of mammalian cells in culture. Transient expression of a luciferase reporter gene was observed. Under selective conditions, stable expression of a neomycin gene was established as a result of integration into the genome. HMG1-mediated gene delivery was as efficient as calcium phosphate-mediated transfection but without associated cell damage. In addition, stable transfectants obtained after selection for G418 resistance usually integrated only one copy of the transfected DNA in contrast to the high unpredictable number obtained by the calcium phosphate method. HMG1 transfection complexes were not toxic to cultured cells, even at high concentrations.

Amino Acid Sequence↗
PubMed1996

A recombinant, arginine-glycine-aspartic acid (RGD) motif from foot-and-mouth disease virus binds mammalian cells through vitronectin and, to a lower extent, fibronectin receptors.

The cell-binding abilities of a recombinant, RGD-containing peptide from foot-and-mouth disease virus (FMDV) have been characterized in HeLa and BHK cells. This peptide represents the aa sequence of the solvent-exposed G-H loop of protein VP1 which is involved in cell recognition and infection. The efficiency of the viral motif in promoting cell attachment and spreading is comparable to that shown by fibronectin or vitronectin. Cell binding is inhibited by a monoclonal antibody directed against a viral, RGD-involving B-cell epitope and also by sera against vitronectin (alpha V beta 3/beta 5) and fibronectin (alpha 5 beta 1) receptors. In addition, a synthetic RGD peptide, which is a ligand for both integrins, prevents the cell binding mediated by the FMDV domain. These data demonstrate that the FMDV RGD motif is a potent ligand for cell-receptor integrins and sufficient to promote cell attachment to susceptible cells mainly through the vitronectin receptor.

Amino Acid Sequence↗
PubMed1996

Synthesis of a modified gene encoding human ornithine transcarbamylase for expression in mammalian mitochondrial and universal translation systems: a novel approach towards correction of a genetic defect.

The mitochondrial (MT) genome is a potential means of gene delivery to human cells for therapeutic expression. As a first step towards this, we have synthesized a gene coding for mature human ornithine transcarbamylase (OTC) by recursive PCR using 18 oligodeoxyribonucleotides, each 70-80 nucleotides in length, using codons which should allow translation in accordance with both mammalian mt and universal codon usage. Flanking mt DNA sequences were incorporated which are designed to facilitate site-specific cloning into the mt genome. Expression of this human gene in Escherichia coli leads to an immunoreactive OTC product of the correct size and N-terminal amino-acid sequence, but which forms inclusion bodies and lacks enzymatic activity.

Amino Acid Sequence↗
PubMed1996

Liposomes and viruses for gene therapy of cystic fibrosis.

Cystic fibrosis (CF) is a common, life-threatening autosomal recessive disease caused by mutations in the CFTR gene. It affects the function of the lung, gut, and liver. Present strategies for CF aim to correct the defect by introducing a normal copy of the CFTR gene into affected epithelial cells. Two vector systems have been proposed for gene therapy trials, replication defective adenovirus and cationic liposome/DNA complexes. Adenoviral vectors have been used in Phase I trials and in most cases give transient molecular and/or electrophysiological restitution of the ion transport cellular defects of CF. However, a dose of 10(9) pfu/ml applied to the lung led, in one patient, to a transient inflammatory reaction. New adenoviral vectors are presently being developed to solve this problem. Our studies using liposome/DNA complexes to deliver CFTR cDNA to the nasal epithelium were carried out in the double blind trial and showed no treatment-related local or general adverse reactions and significant small but transient correction of the ion transport defect. The results for both current approaches demonstrate the need for substantial improvement of the efficiency and duration of transgene expression to reach therapeutically relevant levels.

Adenoviridae↗
PubMed1996

Fetal somatic gene therapy.

Fetal somatic gene therapy is emerging as a new experimental approach, in particular to prevent irreversible perinatal disease manifestation for many inherited conditions. Early therapeutic gene application may also allow targeting of still expanding stem cell populations of organ or cell systems inaccessible later in life and help to avoid immune sensitization against the therapeutic vector system or transgene protein product. The progress in development of ultrasound scanning and embryofetoscopy over the last decade has made minimally invasive administration of therapeutic gene transfer vectors to the fetus in utero possible in principle. We review here the different considerations in choosing candidate diseases, the possible routes of administration and times in fetal development for application of a therapeutic gene and discuss the benefits and problems of present vector systems in this context. Given the many unknown aspects of fetal gene transfer, it is essential to extensively investigate this new approach to gene therapy in animal models for specific diseases, to improve on the technology of delivery and to assess efficacy of expression as well as the possible side effects before application to humans can be considered.

Adenosine Deaminase↗
PubMed1996

Effect of amniotic fluid on cationic lipid mediated transfection and retroviral infection.

In preparation for foetal gene therapy by intra-amniotic gene application, we have investigated the effect of amniotic fluid on several gene transfer systems. In vitro lipofection of embryonically derived 3T3 cells by several of the tested cationic lipids decreases in the presence of human amniotic fluid, while two formulations, Lipid 67 and Tfx-50, remain highly active. As some body fluids are known to inactivate most retroviral vectors, we investigated the influence of amniotic fluid on the efficiency of infection of 3T3 cells by murine leukaemia virus (MoMLV)-based vectors, including amphotropic and ecotropic retrovirus, and a vesicular stomatitis virus G (VSV-G) glycoprotein pseudotyped retroviral vector. All showed a decrease of infectivity from 21 to 56% in the presence of amniotic fluid. The ecotropic retrovirus is the most infectious under normal conditions as well as in amniotic fluid. Our results suggest that intra-amniotic injection may allow efficient gene transfer using either amniotic fluid-resistant cationic lipids or ecotropic retroviral vectors in a murine in vivo model for foetal gene therapy. The VSV-G-pseudotyped vector, although displaying a decrease of infectivity, remains of great interest for gene delivery, because of its broad host range and because of the high virus titers achievable. Finally, a baculovirus-based vector shows no decrease of its infectivity and does not seem to be affected by amniotic fluid but has only low infectivity on the tested foetal fibroblast cell line.

3T3 Cells↗
PubMed1995

Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.

We report the results of a double-blind, placebo-controlled trial in nine cystic fibrosis (CF) subjects receiving cationic liposome complexed with a complementary DNA encoding the CF transmembrane conductance regulator (CFTR), and six CF subjects receiving only liposome to the nasal epithelium. No adverse clinical effects were seen and nasal biopsies showed no histological or immuno-histological changes. A partial restoration of the deficit between CF and non-CF subjects of 20% was seen for the response to low Cl- perfusion following CFTR cDNA administration. This was maximal around day three and had reverted to pretreatment values by day seven. In some cases the response to low Cl- was within the range for non-CF subjects. Plasmid DNA and transgene-derived RNA were detected in the majority of treated subjects. Although these data are encouraging, it is likely that transfection efficiency and the duration of expression will need to be increased for therapeutic benefit.

Base Sequence↗
PubMed1995

The challenge of fetal gene therapy.

Three recent reports describe the first in vivo attempts at fetal gene therapy. The results underline the need for more intensive studies of the scientific and ethical implications of this new and perhaps more preventive approach to gene therapy.

Animals↗
PubMed1995

The introduction of two silent mutations into a CFTR cDNA construct allows improved detection of exogenous mRNA in gene transfer experiments.

Phase one clinical trials for gene therapy of cystic fibrosis are in progress using either liposomes or adenoviral vectors for CFTR gene transfer to epithelial cells in the airways. In addition to electrophysiological measurements, expression of vector CFTR is usually assessed by RT-PCR. We have developed a CFTR-expression vector, pCFAS, that simplifies the distinction of transgene-derived CFTR mRNA from endogenous mRNA. Two point mutations were introduced into CFTR cDNA which eliminated a SphI restriction site and created a new, unique AgeI restriction site. Neither mutation altered the predicted amino acid sequence of the protein. Restriction digestion of RT-PCR products from cells transfected with pCFAS allowed the differentiation of transgene and endogenous CFTR transcripts. To verify function of the mutated CFTR, the plasmid was transferred into freshly obtained nasal epithelial cells from CF patients ex vivo using cationic liposomes. Fluorescence microscopy using the halide-sensitive fluorophore SPQ demonstrated restoration of cAMP-mediated Cl- secretion. This plasmid will be useful for CFTR gene transfer studies in vitro and in vivo.

Base Sequence↗
PubMed1995

In vitro liposome-mediated DNA transfection of epithelial cell lines using the cationic liposome DC-Chol/DOPE.

Clinical trials using cationic liposome-mediated DNA transfer have now been initiated for several disorders including cystic fibrosis. Previous studies have shown that the level of gene expression achieved may be dependent on the formulation of the DNA-liposome complex and the cell type transfected. We have investigated, in vitro, the effect of parameters such as DNA:liposome ratio, dose and concentration on the level of transgene expression in epithelial cell lines using the cationic liposome DC-Chol/1,2-dioleoyl phosphatidylethanolamine (DOPE). A narrow range of conditions was found to produce maximal level of transgene expression within a particular cell line, as detected using the reporter molecule beta-galactosidase (beta-gal). beta-Gal expression was significantly enhanced by formulation of the DNA-DC-Chol/DOPE complexes in physiological solution at pH 9.0. Under standard in vitro transfection conditions, increased incubation time of the DNA-liposome complexes with cells resulted in increased transgene expression. In contrast, at relatively high DNA and liposome dose and concentrations, beta-gal activity was maximal after only 1 h of incubation, with a subsequent decrease in expression with time. The maximum level of expression that could be produced using fully optimised transfection conditions, however, was still highly dependent on each cell type analysed. Correlation of these findings with similar studies in vivo are now critical to determine the optimal formulation of DNA-liposome complexes for clinical application.

Animals↗
PubMed1995

Gene delivery and expression mediated by an integrin-binding peptide.

The ability to transfer sufficient DNA to specific target cells remains one of the main limitations to the development of gene therapy. For this reason much attention is being paid to the development of new gene delivery systems, both viral and non-viral. We describe gene transfer with a polycation-DNA complex which contains an integrin-binding domain. Integrin-mediated gene delivery has several potential advantages. Such complexes are less likely than other receptor-mediated gene delivery complexes to be constrained by the size of the complex. The ligands are small peptides, resembling naturally occurring integrin ligands, which minimises the possibility of complexes inducing an immune response in vivo.

Amino Acid Sequence↗