Fetal somatic gene therapy--a preventive approach to the treatment of genetic disease: the case for.
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Disease Models, Animal↗
Biomedical subjects
C Coutelle
Publications and source records attributed to C Coutelle.
[Gene therapy of cystic fibrosis; review of completed clinical studies].
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Adenoviridae↗
A novel human suspension culture packaging cell line for production of high-titre retroviral vectors.
Retroviruses are currently the most widely used vectors in clinical trials for gene therapy. These vectors are, however, limited by low titres partly due to the restrictive nature of monolayer cell culture. We have developed a stable suspension producer cell line derived from human lymphoblastoid cells (WIL-2) by electroporating these cells with the necessary trans components required for production of defective retrovirus particles which encode a nuclear localising beta-galactosidase gene. We show that this anchorage-independent cell line generates viruses at a titre of 7 x 10(5) iu/ml on NIH3T3 indicator cells which remains constant after at least 2 months in culture. The producer cells can be cultured at a density of 6 x 10(6) cells/ml with consistent virus titre production. WIL-2 can also be grown as single cells by rotation culture while maintaining virus production. By treating the cells with the transcriptional activator sodium butyrate titres above 1 x 10(6) i.u./ml are achieved. Concentrating viral supernatants by ultrafiltration can further increase virus titre to 5 x 10(8) i.u./ml. Even at these high titres no replication-competent virus was detected. Virus titre fell only slightly when cells were placed in serum-free media before harvest. The generation of this novel cell line provides proof-of-principle that large-scale production of retroviral vectors in serum-free growth conditions can be safely generated for use in gene therapy.
Blotting, Western↗
The nuclear pore complex is involved in nuclear transfer of plasmid DNA condensed with an oligolysine-RGD peptide containing nuclear localisation properties.
One of the major barriers to efficient gene transfer and expression of nonviral vectors for gene therapy is passage across the nuclear envelope. We have previously shown that an oligolysine-RGD peptide that condenses plasmid DNA and binds to cell surface integrins can mediate increased internalisation of plasmid DNA into cells and synergistic enhancement of gene expression when complexed to a cationic lipid. In this report, we show that this enhancement is due to increased nuclear transfer of the plasmid DNA. We have applied the digitonin-permeabilised cell system that has been well established for the study of the nuclear transport of proteins to examine the nuclear transfer of plasmid DNA. Nuclear transfer of plasmid DNA complexed to an oligolysine-RGD peptide and lipofectamine appears to be an energy-dependent process involving the nuclear pore complex, since it is inhibited at 4 degrees C and by treatment with wheat germ agglutinin or with an antibody to the nuclear pore complex which all block nuclear protein import. In accordance with active nuclear transport, we have shown that all these treatments inhibit expression of a luciferase reporter plasmid in permeabilised cells. Nuclear transfer of pDNA is enhanced in mitotic cells, but cell division is not a prerequisite for transfer. We propose that the oligolysine-RGD peptide acts as a nuclear localisation signal and that the cationic lipid is more important for cell entry and endosome destabilisation than nuclear transfer.
Active Transport, Cell Nucleus↗
Perspectives on gene therapy for cystic fibrosis airway disease.
Since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene nearly 12 years ago, cystic fibrosis (CF) has become one of the most intensively investigated monogenetic disorders considered approachable by gene therapy. This has resulted in over 20 clinical trials currently under way, concluded or awaiting approval. Despite the initial promise of gene therapy for CF, and the demonstration of successful gene transfer to the nose and airways of individuals, it has not so far been as effective as initially projected. Here we discuss the rationale behind CF gene therapy and dissect the vast array of literature representing the work that ultimately brought about the current phase I/II clinical trials. In the context of human trials, we review the limitations of current vector systems for CF gene therapy. We come to the conclusion that at present none of the application methods and vector systems are able to achieve the level and persistence of CFTR gene expression in the affected epithelia of CF patients that is required for therapeutic success. We also outline the challenges that must be overcome and describe some of the novel approaches to be taken in order to attain the curative therapy that was originally envisaged for this disease.
Adenoviridae↗
Metabolic gene polymorphism frequencies in control populations.
Using the International Project on Genetic Susceptibility to Environmental Carcinogens (GSEC) database containing information on over 15,000 control (noncancer) subjects, the allele and genotype frequencies for many of the more commonly studied metabolic genes (CYP1A1, CYP2E1, CYP2D6, GSTM1, GSTT1, NAT2, GSTP, and EPHX) in the human population were determined. Major and significant differences in these frequencies were observed between Caucasians (n = 12,525), Asians (n = 2,136), and Africans and African Americans (n = 996), and some, but much less, heterogeneity was observed within Caucasian populations from different countries. No differences in allele frequencies were seen by age, sex, or type of controls (hospital patients versus population controls). No examples of linkage disequilibrium between the different loci were detected based on comparison of observed and expected frequencies for combinations of specific alleles.
Black People↗
Role of alcohol dehydrogenase 3 and cytochrome P-4502E1 genotypes in susceptibility to cancers of the upper aerodigestive tract.
Alcohol is a recognized risk factor for upper aerodigestive tract (UAT) cancers, but the mechanism by which alcohol causes cancer remains obscure. Ethanol is oxidized to acetaldehyde (the suspected carcinogenic agent in alcohol) by alcohol dehydrogenases (ADHs) and cytochrome P-4502E1 (CYP2E1), both of which exhibit great inter-individual variability in activity. The hypothesis that these polymorphisms influence susceptibility to alcohol-related cancers remains poorly documented. We investigated whether ADH(3) and CYP2E1 DraI and RsaI genotypes modified the risk of UAT cancers among 121 oral cavity/pharyngeal cancer patients, 129 laryngeal cancer patients, and 172 controls, all French Caucasians. Cancer risks and gene-alcohol interactions were analyzed by unconditional logistic regression, accounting for potential confounders. ADH(3) genotype was not associated with UAT cancer. In contrast, a 2-fold risk of oral cavity/pharyngeal (OR = 2.0, 95% CI 1.0-3.9) and laryngeal (OR = 1.8, 95% CI 1.0-3.5) cancers was observed for carriers of the CYP2E1 DraI C variant allele compared with other individuals. The risk associated with the CYP2E1 RsaI c2 variant allele also increased for oral cavity/pharyngeal cancer (OR = 2.6, 95% CI 1.0-6. 6). The effects of ADH(3) or CYP2E1 genotype and alcohol or tobacco were independent. The highest risk of oral cavity/pharyngeal cancer was observed among the heaviest drinkers (>80 g/day) with the CYP2E1 DraI C allele (OR = 5.8, 95% CI 1.9-18.2) or the CYP2E1 RsaI c2 allele (OR = 7.2, 95% CI 1.4-38.2) compared with lighter drinkers with other genotypes. Our study suggests that CYP2E1 genotype modifies the risk of UAT cancers, but due to the low frequency of CYP2E1 variant alleles, large-scale studies are needed to confirm our findings.
Alcohol Drinking↗
Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression.
A recombinant, multifunctional protein has been designed for optimized, cell-targeted DNA delivery and gene expression in mammalian cells. This hybrid construct comprises a viral peptide ligand for integrin alpha(V)beta(3) binding, a DNA-condensing poly-L-lysine domain, and a complete, functional beta-galactosidase protein that serves simultaneously as purification tag and DNA-shielding agent. This recombinant protein is stable; it has been produced successfully in Escherichia coli and can be purified in a single step by affinity chromatography. At optimal molar ratios, mixtures of this vector and a luciferase-reporter plasmid form stable complexes that transfect cultured cells. After exposure to these cell-targeted complexes, steady levels of gene expression are observed for more than 3 days after transfection, representing between 20 and 40% of those achieved with untargeted, lipid-based DNA-condensing agents. The principle to include viral motifs for cell infection in single polypeptide recombinant proteins represents a promising approach towards the design of non-viral modular DNA transfer vectors that conserve the cell-target- ing specificity of native viruses and that do not need further processing after bioproduction in a recombinant host.
Amino Acid Sequence↗
Introduction of chloramphenicol resistance into the modified mouse mitochondrial genome: cloning of unstable sequences by passage through yeast.
Despite increasing awareness of the importance of the mitochondrial genome in human pathology, very few attempts have been made so far toward genetic engineering of mitochondrial DNA (mtDNA). One of the reasons for this slow progress is the difficulty of cloning mtDNA in Escherichia coli, a trait in common with repetitive or palindromic sequences, and some viral sequences. We have previously made a construct containing the entire mouse mitochondrial genome and a cDNA sequence coding for human ornithine transcarbamylase in a yeast/bacterial shuttle vector, which can be stably maintained in E. coli. We wished to modify this vector for mitochondrial gene therapy by the addition of mitochondrial chloramphenicol resistance, conferred by a point mutation in the 16S rRNA gene. Attempts to modify this construct by a straightforward cloning approach in E. coli proved unsuccessful. Two successful strategies for modification of large unstable constructs in both E. coli and the yeast Saccharomyces cerevisiae are compared here.
Animals↗
Synergistic growth factors enhance rat liver proliferation and enable retroviral gene transfer via a peripheral vein.
BACKGROUND & AIMS: Genetic diseases reflecting abnormal hepatocyte function are potentially curable through gene therapy. Retroviral vectors offer the potential for permanent correction of such conditions. These vectors generally require cell division to occur to allow provirus entry into the nucleus, initiated in many experimental protocols by partial hepatectomy. We have explored methods to improve the efficiency of retroviral gene transfer that avoid the need for liver damage. METHODS: Triiodothyronine (T3) and keratinocyte growth factor (KGF) were used to induce hepatic proliferation in rats. The effects of intraportal and peripheral administration of a modified retrovirus that encoded the Lac Z gene during growth factor-induced liver hyperplasia were analyzed. RESULTS: T3 initiated hepatocyte proliferation midzonally; after KGF, proliferation was more diffuse. Optimal concentrations of T3 and KGF acted synergistically to induce proliferation in 61% of hepatocytes in the intact liver. This enabled in vivo hepatocyte transduction, leading to gene expression by up to 7.3% of hepatocytes after intraportal retroviral vector administration and 7. 1% after peripheral venous administration. CONCLUSIONS: T3 and KGF act synergistically to induce hepatocyte proliferation in undamaged liver. The liver can be simply transduced with integrating vectors via the peripheral venous system during a wave of growth factor-induced proliferation.
Animals↗
Cell delivery, intracellular trafficking and expression of an integrin-mediated gene transfer vector in tracheal epithelial cells.
The mechanism of cell entry and intracellular fate of a gene transfer vector composed of a receptor-targeting, DNA-condensing peptide, RGD-oligolysine, a luciferase encoding plasmid DNA (pDNA) and a cationic liposome was examined. We demonstrate by confocal microscopy, electron microscopy and subcellular fractionation that the major mechanism of entry of the vector is endocytic. The vector complex rapidly (5 min) internalizes into early endosomes, then late endosomes and lysosomes. Entry involves, at least in part, clathrin-coated pit-mediated endocytosis since different conditions or drugs known to influence this pathway modify both uptake of pDNA and its expression. The observed increase in expression with addition of a lip some correlated with an increase in the rate of transfer of the pDNA to lysosomes, a decrease in intracellular recycling and exocytosis of the pDNA and an increase in the amount of pDNA in the nuclear fraction. Trafficking within the cell involved endosome fusion and the acid environment of the endosomes-lysosomes was beneficial for expression. After 30 min both the peptide and pDNA localized to the nucleus and the amount of intact pDNA in the nuclear fraction was highest with liposome and peptide. A better understanding of the cellular mechanisms by which vectors transfer to and traffic in cells should help design improved vectors.
Cation Exchange Resins↗
Tri-iodothyronine and a deleted form of hepatocyte growth factor act synergistically to enhance liver proliferation and enable in vivo retroviral gene transfer via the peripheral venous system.
Retroviral vectors integrate into the target cell genome in a stable manner and therefore offer the potential for permanent correction of the genetic diseases that affect the liver. These vectors, however, usually require cell division to occur in order to allow provirus entry into the nucleus. We have explored clinically acceptable methods to improve the efficiency of retroviral gene transfer to the liver, which avoid the need for liver damage. Tri-iodothyronine (T3) and recombinant hepatocyte growth factor have previously been used to induce hepatocyte proliferation in rat livers and allow in vivo retroviral gene transfer. We investigated the combined effects of these growth factors, with their differing mechanisms of action, on hepatocyte proliferation in vivo and assessed their effectiveness in priming cells for retroviral gene transfer. During the phase of hepatocyte proliferation retrovirus was administered via either the portal or tail vein. Acting synergistically, T3 and a truncated form of recombinant hepatocyte growth factor (dHGF) induced 30% of hepatocytes in normal rat liver to enter DNA synthesis at 24 h. This increased proliferation enabled the liver to be transduced in vivo by retroviral vectors via either the portal or peripheral venous system, achieving transduction efficiencies of 6.9 +/- 1.6% and 4.3 +/- 0.4% respectively. Thus, the liver can be simply and conveniently transduced in vivo with integrating vectors, introduced via the peripheral venous system during a wave of growth factor-induced proliferation, pointing the way to clinically applicable gene transfer techniques.
Animals↗
Selective in vivo transfection of murine biliary epithelia using polycation-enhanced adenovirus.
We have investigated the use of polycations to increase adenovirus-mediated expression of transgenic protein to the biliary epithelia with a view to gene therapy for hepatobiliary disease in CF. We have shown that adenovirus carrying the beta-galactosidase transgene transfect both human and mouse biliary epithelia in primary culture and that in both instances adenovirus transfection can be significantly increased by co-complexing with polycation. In vivo administration of 1 x 109 p.f.u. adenovirus co-complexed with the polyamine polyethyenimine (PEI) into the mouse biliary duct leads to >80% positively stained biliary epithelia while adenovirus alone at the same titre infected <5% biliary epithelia. We suggest that the use of low titre polycation enhanced adenoviral delivery to the biliary tree of CF patients could be of therapeutic significance. As a prelude to an extensive in vivo functional investigation in CF null mice we have shown that Ad5/polycation complexes deliver functional CFTR to non-CFTR expressing cells in vitro more efficiently than Ad5 alone.
Adenoviridae↗
Haemoglobin interferes with the ex vivo luciferase luminescence assay: consequence for detection of luciferase reporter gene expression in vivo.
The luciferase reporter gene is a useful tool for determining the efficacy of transfection of plasmid DNA and adenovirus-mediated gene transfer in vivo. However, we report here that the haemoglobin present in tissue samples can mask the detection of the luciferase activity and lead to underestimation of the luciferase levels. We evaluated the degree of interference in different organ samples of mice and investigated the possibilities for removal of haemoglobin from tissue samples by: (1) perfusion of the whole animal; (2) different hypotonic treatments lysing preferentially red blood cells; and (3) chromatographic separation. Removal of haemoglobin resulted in significantly improved detection of luciferase activity from tissue samples. The results indicate that the luciferase activity determined in tissue samples may not reflect the actual level of reporter gene expression, if contaminating blood is not taken into consideration.
Adenoviridae↗
Retargeting of adenoviral vectors to neurons using the Hc fragment of tetanus toxin.
The Hc fragment of tetanus toxin (Hc) retains the specific nerve cell binding and transport properties of the holotoxin, but lacks any toxicity. We are investigating the potential for utilising its neurotropism for targeted gene delivery to the central nervous system. Previously we reported the use of Hc-polylysine conjugates for selective gene transfer into neuronal cells in vitro. However, as attempts to apply these constructs in vivo were not successful, we have extended these studies to modification of the tropism of adenoviral vectors. Either Hc-polylysine conjugates or the Fab fragment of a neutralising anti-knob antibody covalently bound to Hc were attached to the virus. Infection of neuronal and non-neuronal cell lines with retargeted virus showed highly increased neuronal cell selectivity, but no significant enhancement of gene delivery into these cells. High concentrations of free Hc blocked the infectivity of the retargeted vector efficiently. Intramuscular injection of retargeted virus into mouse tongues resulted in selective gene transfer to the neurons of the hypoglossal nucleus, where no pathological changes were observed. As differentiated neurons do not undergo cell division, appropriate vectors carrying a thymidine kinase gene, which allows selective elimination of dividing cells, may be exploitable for the treatment of tumours of the central nervous system. The demonstrated suitability of the Hc fragment of tetanus toxin as targeting moiety for viral vectors also indicates a potential for gene therapy of inherited neurodegenerative diseases such as spinal muscular atrophy.
Adenoviridae↗
Genetic polymorphism of alcohol dehydrogenase in europeans: the ADH2*2 allele decreases the risk for alcoholism and is associated with ADH3*1.
Polymorphism at the ADH2 and ADH3 loci of alcohol dehydrogenase (ADH) has been shown to have an effect on the predisposition to alcoholism in Asian individuals. However, the results are not conclusive for white individuals. We have analyzed the ADH genotype of 876 white individuals from Spain (n = 251), France (n = 160), Germany (n = 184), Sweden (n = 88), and Poland (n = 193). Peripheral blood samples from healthy controls and groups of patients with viral cirrhosis and alcohol-induced cirrhosis, as well as alcoholics with no liver disease, were collected on filter paper. Genotyping of the ADH2 and ADH3 loci was performed using polymerase chain reaction-restriction fragment length polymorphism methods on white cell DNA. In healthy controls, ADH2*2 frequencies ranged from 0% (France) to 5.4% (Spain), whereas ADH3*1 frequencies ranged from 47. 6% (Germany) to 62.5% (Sweden). Statistically significant differences were not found, however, between controls from different countries, nor between patients with alcoholism and/or liver disease. When all individuals were grouped in nonalcoholics (n = 451) and alcoholics (n = 425), ADH2*2 frequency was higher in nonalcoholics (3.8%) than in alcoholics (1.3%) (P =.0016), whereas the ADH3 alleles did not show differences. Linkage disequilibrium was found between ADH2 and ADH3, resulting in an association of the alleles ADH2*2 and ADH3*1, both coding for the most active enzymatic forms. In conclusion, the ADH2*2 allele decreases the risk for alcoholism, whereas the ADH2*2 and ADH3*1 alleles are found to be associated in the European population.
Adult↗
Targeted gene delivery into alpha9beta1-integrin-displaying cells by a synthetic peptide.
We have investigated the usefulness of two small synthetic peptides comprising either a linear or a cyclic PLAEIDGIEL domain and a DNA-binding moiety of 16 lysine residues to mediate gene transfer selectively into alpha9beta1-integrin-displaying cells. Such specific gene delivery could only be achieved with the peptide containing the cyclic PLAEIDGIEL domain. However, inclusion of the cationic liposome Lipofect-AMINE into the peptide/DNA complexes resulted for both peptides in efficient gene transfer with significant targeting specificity. Naturally, the integrin alpha9beta1 is present only in a few highly specialised tissues and abundant throughout the human airway epithelia in vivo. Targeting gene vectors to this integrin therefore appears a useful approach to gene therapy of lung diseases such as cystic fibrosis. As the integrin alpha9beta1 is associated with tissue differentiation during foetal development and may cause resurgence of the foetal phenotype in colon cancers, such vectors may also be applicable for prenatal and cancer gene therapy.
Cation Exchange Resins↗
Calcium ions as efficient cofactor of polycation-mediated gene transfer.
We investigated the effect of calcium on the transfection of non-viral DNA transfer systems. Cationic proteins such as the nuclear protein H1, the polycation polylysine and a number of commercial transfection agents exhibited high transfection rates in the presence of Ca2+. Without Ca2+ H1 and HMG1 were inactive in transfection of the human permanent endothelial cell line ECV 304 while cationic liposomes such as Lipofectin and Lipofectamine did not show any Ca2+ dependence. More detailed experiments showed that Ca2+ was replaceable by the lysosomotropic agent chloroquine. Furthermore, it was possible to separate the transfection-enhancing role of Ca2+ from the actual transfection process by adding Ca2+ to the cells after the transfection period and still to obtain a significant transgene expression. This makes it possible to distinguish between cellular uptake of H1 (or mediator)-DNA complexes and endocytotic release. We also replaced soluble Ca2+ by Ca-phosphate precipitates not containing DNA and obtained similar transfection results. This allowed us to suggest that the addition of free Ca2+ to the transfection medium resulted in nascent Ca-phosphate microprecipitates. The known fusogenic and membranolytic activity of such microprecipitates could facilitate the transport through and the release of the transfecting complexes from the endosomal/lysosomal compartment.
Calcimycin↗