In utero gene therapy: the case for.
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Clinical Trials as Topic↗
Biomedical subjects
C Coutelle
Publications and source records attributed to C Coutelle.
Successful expression of beta-galactosidase and factor IX transgenes in fetal and neonatal sheep after ultrasound-guided percutaneous adenovirus vector administration into the umbilical vein.
In utero somatic gene therapy in the later stages of pregnancy may allow targeting of organ systems which are difficult to reach later in life and to prevent the development of tissue damage otherwise caused by the early onset of inherited diseases. We report here on the percutaneous delivery of two adenoviral vectors, containing the beta-galactosidase reporter gene and the human Factor IX gene respectively, to the fetal liver and circulation by ultrasound-guided umbilical vein puncture similar to procedures used in human pregnancy. Vector spread, as detected by PCR analysis for the beta-galactosidase encoding vector, was found in almost all fetal and neonatal organs and in the maternal liver. Expression of the beta-galactosidase transgene was detected in many fetal tissues by RT-PCR. High beta-galactosidase production was shown by immuno-histochemistry predominantly in the liver, where about 30percent of the hepatocytes stained positive, and in the adrenal cortex. Production of factor IX was determined by ELISA in the plasma of treated fetuses and newborn lambs and reached at birth up to 80percent of the normal human plasma concentration. This demonstrates a very hopeful proof of principle for the development of prenatal treatment of many genetic diseases but also requires more detailed investigations with respect to the observed systemic spread of the vector.
Adenoviridae↗
Tipping the scales in favour of mitochondrial gene therapy.
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Amino Acid Substitution↗
Non-viral neuronal gene delivery mediated by the HC fragment of tetanus toxin.
Many inherited neurological diseases and cancers could potentially benefit from efficient targeted gene delivery to neurons of the central nervous system. The nontoxic fragment C (HC) of tetanus toxin retains the specific nerve cell binding and transport properties of tetanus holotoxin. The HC fragment has previously been used to promote the uptake of attached proteins such as horseradish peroxidase, beta-galactosidase and superoxide dismutase into neuronal cells in vitro and in vivo. We report the use of purified recombinant HC fragment produced in yeast and covalently bound to polylysine [poly(K)] to enable binding of DNA. We demonstrate that when used to transfect cells, this construct results in nonviral gene delivery and marker gene expression in vitro in N18 RE 105 cells (a neuroblastoma x glioma mouse/rat hybrid cell line) and F98 (a glioma cell line). Transfection was dependent on HC and was neuronal cell type specific. HC may prove a useful targeting ligand for future neuronal gene therapy.
Cation Exchange Resins↗
A cell adhesion peptide from foot-and-mouth disease virus can direct cell targeted delivery of a functional enzyme.
The G-H loop of foot-and-mouth disease virus is a disordered protrusion of the VP1 protein exposed on the virion surface. This short stretch includes an arginine-glycine-aspartic acid tripeptide, a recognized integrin-binding motif, which is responsible for cell attachment and infection. Eight copies of a peptide reproducing the amino acid sequence of this FMDV ligand have been displayed in solvent-exposed regions on an enzymatically active recombinant beta-galactosidase. This viral peptide segment enables the chimeric enzyme to bind mammalian cell lines with different efficiencies, probably depending on the number of suitable cell receptors present on each of them. Moreover, it also promotes the internalization of the attached enzyme, which is transiently active inside the cells. These results suggest further exploration of the potential use of short adhesion peptides of viral origin as cell attachment tags to direct the targeted delivery of both genes and enzymes, instead of whole, infectious viruses.
Amino Acid Sequence↗
Re: Alcohol dehydrogenase 3 genotype and risk of oral cavity and pharyngeal cancers.
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Alcohol Dehydrogenase↗
A novel peptide, PLAEIDGIELTY, for the targeting of alpha9beta1-integrins.
Targeting gene therapy vectors to abundant receptors on airway epithelia may allow a significant enhancement of gene delivery and thereby be of particular importance for the gene therapy of cystic fibrosis. Alpha9beta1-integrins are highly expressed throughout the human airway epithelia in vivo, irrespective of any particular clinical status. Aiming to improve the targeting of our non-viral integrin-mediated gene transfer systems to airway epithelia, we searched for a short tenascin C-derived peptide which would bind to these integrins. By utilizing recombinant bacteriophages that display overlapping regions of the third fibronectin type III repeat of tenascin C (TNfn3), we were able to localize its alpha9beta1-integrin binding site to the B-C loop of TNfn3. A synthetic Pro-Leu-Ala-Glu-Ile-Asp-Gly-Ile-Glu-Leu-Thr-Tyr peptide (PLAEIDGIELTY) was shown to displace alpha9beta1-integrin-expressing cells completely from binding to TNfn3. This peptide, therefore, may prove useful both for the examination of the functional importance of alpha9beta1-integrins in vivo and the development of gene therapy vectors or drugs targeting these integrins.
Amino Acid Sequence↗
An RGD-oligolysine peptide: a prototype construct for integrin-mediated gene delivery.
We have synthesized a linear, bifunctional peptide that comprises an integrin-targeting domain containing an arginine-glycine-aspartic acid tripeptide motif and a DNA-binding moiety consisting of a short stretch of 16 lysine residues. This peptide can form distinctive, condensed complexes with DNA and is capable of mediating its delivery and expression in a variety of mammalian cells in culture. Internalization is mediated by cell surface integrin receptors via a mechanism that is known to be phagocytic. We have analyzed the relationship between DNA and peptide and have investigated the conditions suitable for optimal gene delivery. The formation of condensed peptide DNA complexes leads to resistance to nuclease degradation. The level of reporter gene expression obtained is dependent on the peptide-to-DNA ratio and is enhanced in the presence of the endosomal buffer chloroquine, polyethyleneimine, and deactivated adenovirus during gene delivery. Under optimal conditions the levels of reporter gene expression obtained approach or even exceed those obtained with DNA delivered with the commercial liposome Lipofectamine. The ability to produce an efficient gene delivery system using small, easily modified, and well-defined constructs that have no constraint of particle size demonstrates the advantages of integrin-targeting peptides for gene transfer.
3T3 Cells↗
Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector.
Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.
Amino Acid Sequence↗
Towards gene therapy of mitochondrial disorders.
Mitochondrial disorders are characterized by protein deficiencies affecting the structure and function of mitochondria. The protein deficiencies are caused by mutations either in a nuclear gene or in the mitochondrial genome. Most current approaches to gene therapy of mitochondrial diseases aim at expression of the corrective gene sequence by nuclear/cytoplasmic expression. However, the mitochondrial genome and its autonomous expression system offer the potential of an alternative gene therapy strategy: the introduction of nuclear gene sequences into the mitochondrial genome and their expression by the mitochondrial gene expression system. In addition to its potential for gene therapy, the introduction and expression of an exogenous gene in mitochondria would provide an invaluable tool towards the understanding of mitochondrial genome expression and its regulation.
Animals↗
Retroviral gene transfer to the liver in vivo during tri-iodothyronine induced hyperplasia.
The liver is an important target organ for gene therapy but its mitotic quiescence makes it resistant to integrative gene transfer. Retrovirus-based vectors integrate into liver cells in vivo but require the liver to be primed before transduction; experimentally a 70% hepatectomy is commonly used to stimulate regeneration, rendering the liver susceptible to transduction during the resulting wave of cell proliferation. Our aim was to develop a clinically acceptable method of inducing hepatocyte replication before in vivo retroviral gene transfer which is both simple and effective. We have used the physiological hormone tri-iodothyronine (T3) to stimulate hepatocyte replication. A single dose of T3 (400 micrograms/100 g bw) was given subcutaneously to euthyroid rats. This produced a labelling index of 31.7% in the hepatocyte population without histological or biochemical evidence of preceding liver damage. Following T3 administration the rat livers were transfected in vivo with an amphotropic retrovirus, TELCeB/AF-7 which encodes the beta-galactosidase reporter gene together with a nuclear localisation signal. Transgene expression was noted only within the liver where 1.3% of hepatocytes expressed the beta-galactosidase enzyme. This compared to 5.2% of hepatocytes transduced following a 70% hepatectomy, and 0.02% in animals receiving neither T3 nor partial hepatic resection before transduction. T3 administration is a simple way to prime the liver before in vivo retroviral vector-based gene transfer.
Animals↗
Enhanced in vitro and in vivo gene delivery using cationic agent complexed retrovirus vectors.
Retroviruses are, at present, the most efficient integrative vectors available for gene delivery. However, these viruses are still limited by relatively low titres. Although several protocols exist to improve virus titre most of them are time-consuming and unable to provide sufficient virus for in vivo applications. Virus titre can be enhanced by polybrene and other cationic agents. By investigating a broad range of cationic agents for their ability to enhance virus infectivity we found that both ecotropic and amphotropic retrovirus infection could be increased. In particular, the lipopolyamine dioctadecylamidoglycylspermine (DOGS) gave up to one order of magnitude enhancement above polybrene-mediated infection without cytotoxicity. To increase virus infectivity further we combined the enhancing effect of DOGS on virus infectivity with concentration of virus particles by ultrafiltration to reach titres of 1 x 10(9) IU/ml. The in vivo transduction of regenerating rat liver, by an amphotropic retrovirus was increased approximately five-fold by the addition of DOGS compared with virus alone. There was no animal toxicity observed following the administration of DOGS. The improved transduction efficiency seen both in vitro and in vivo following the co-administration of DOGS/virus complexes may be useful for future gene therapy applications.
3T3 Cells↗
Liposomes enhance delivery and expression of an RGD-oligolysine gene transfer vector in human tracheal cells.
Nonviral gene delivery systems consist predominantly of lipoplexes or receptor-targeting and nontargeting polyplexes. We examined integrin-mediated gene delivery using an Arg-Gly-Asp/oligo-L-lysine ([K]16RGD) cyclic peptide and investigated its gene transfer efficiency when associated with a cationic liposome. We demonstrated that human cystic fibrosis and noncystic fibrosis tracheal epithelial cells in culture express integrins that recognise the RGD integrin-binding motif. We found a 10-fold (P < 0.01) increased expression of a luciferase encoding plasmid in these cells when complexing the plasmid to the [K]16RGD peptide as compared with plasmid alone. This increase was specific to the [K]16RGD peptide since neither a [K]16RGE nor a [K]16 peptide gave a comparable increase. Expression was further enhanced 30-fold (P < 0.01) with lipofectamine and the ratio of DNA/peptide/lipofectamine was critical for specificity and expression. Fluorescence and radioactive labelling of the complex showed that the [K]16RGD peptide increased the endocytic uptake of DNA into cells. The cell association of both DNA and peptide increased even further with lipofectamine. Confocal microscopy showed that the [K]16RGD peptide and the DNA internalised together within 30 min and localised to vesicles in the perinuclear region. These results show that an integrin-binding ligand can deliver genetic material to airway cells and that a cationic liposome can enhance the efficacy of this nonviral vector system.
Cation Exchange Resins↗
First pass metabolism of ethanol is strikingly influenced by the speed of gastric emptying.
BACKGROUND: Ethanol undergoes a first pass metabolism (FPM) in the stomach and liver. Gastric FPM of ethanol primarily depends on the activity of gastric alcohol dehydrogenase (ADH). In addition, the speed of gastric emptying (GE) may modulate both gastric and hepatic FPM of ethanol. AIMS: To study the effect of modulation of GE on FPM of ethanol in the stomach and liver. METHODS: Sixteen volunteers (eight men and eight women) received ethanol (0.225 g/kg body weight) orally and intravenously, and the areas under the ethanol concentration time curves were determined to calculate FPM of ethanol. In seven of these subjects, FPM of ethanol was measured after the intravenous administration of 10 mg metoclopramide (MCP) and 20 mg N-butylscopolamine (NBS) in separate experiments to either accelerate or delay GE. GE was monitored sonographically by integration of the antral area of the stomach every five minutes for 90 minutes after oral ethanol intake. In addition, gastric biopsy specimens were taken to determine ADH activity and phenotype, as well as to evaluate gastric histology. Blood was also drawn for ADH genotyping. RESULTS: GE time was significantly delayed by the administration of NBS as compared with controls (p<0.0001) and as compared with the administration of MCP (p<0.0001). This was associated with a significantly enhanced FPM of ethanol with NBS compared with MCP (p = 0.0004). A significant correlation was noted between GE time and FPM of ethanol (r = 0.43, p = 0.0407). Gastric ADH activity did not significantly correlate with FPM of ethanol. CONCLUSION: FPM of ethanol is strikingly modulated by the speed of GE. Delayed GE increases the time of exposure of ethanol to gastric ADH and may therefore increase gastric FPM of ethanol. In addition, hepatic FPM of ethanol may also be enhanced as the result of slower absorption of ethanol from the small intestine. Thus a knowledge of GE time is a major prerequisite for studying FPM of ethanol in humans.
Administration, Oral↗
Modification of the mouse mitochondrial genome by insertion of an exogenous gene.
Using homologous recombination in yeast we have inserted a synthetic gene encoding human ornithine transcarbamylase (sOTC), designed to allow mitochondrial (mt) translation, into the mouse mt genome. Modification of the mt genome was facilitated by its cloning into a yeast centromeric plasmid. The sOTC gene was initially flanked by 25 bp of the mt tRNA(His) gene at its 5' end and by 23 bp of the mt tRNA(Ser (AGY)) gene at its 3' end (Wheeler et al., 1996). In order to achieve homologous recombination the flanking homology was subsequently extended to 525 and 362 bp by the polymerase chain reaction (PCR). The sOTC gene was thus inserted into the cloned mt genome at a unique location between the tRNA(His) and tRNA(Ser (AGY)) genes. Positioning of the sOTC gene between these normally contiguous tRNA genes should allow its processing from the mt polycistronic transcript. The ability to modify the mammalian mt genome in this way is a valuable step towards a functional analysis of mt genetic mechanisms and possibly also towards a gene therapy approach for mt disorders.
Animals↗
Contiguous arrays of satellites 1, 3, and beta form a 1.5-Mb domain on chromosome 22p.
The centromeric heterochromatin of all the human chromosomes is composed of megabases of tandemly repeated satellite DNA. Some of these sequences have been implicated in centromere formation and/or segregation but the arrangement of most of them on a large scale remains largely uncharacterized because of the difficulties in analyzing repetitive DNA. The alpha satellite is the best studied and is present in large tandem arrays at all centromeres, but satellites 1, 3, and beta have also been detected on a number of chromosomes. Here we have used FISH to extended DNA fibers to analyze these satellites on the short arm of the acrocentric chromosome 22. The satellite sequences were found to form a continuous domain spanning about 1.5 Mb and consisting of a major block of satellite 1 flanked by two blocks of beta satellite and three blocks of satellite 3. These six blocks of satellite DNA appear to form contiguous arrays with little intervening DNA.
Centromere↗
Introduction of plasmid DNA into isolated mitochondria by electroporation. A novel approach toward gene correction for mitochondrial disorders.
Mitochondrial disorders are a large group of phenotypically heterogeneous diseases. An understanding of their molecular basis would benefit greatly from the ability to manipulate the mitochondrial genome and/or to introduce functional exogenous DNA into mitochondria. As a first step toward this approach, we have used electroporation to introduce a 7.2-kilobase plasmid DNA into isolated functional mitochondria. Transfer of the DNA at field strengths between 8 and 20 kV/cm was investigated by Southern blot analysis. Maximal plasmid internalization was achieved at a field strength of 14 kV/cm. The functional integrity of the mitochondria after electroporation was verified by enzymatic assays of specific mitochondrial marker enzymes and by measuring respiratory control. At field strengths above 12 kV/cm, an increasing mitochondrial destruction was observed. 12 kV/cm was found to be optimal for the most efficient plasmid internalization while still retaining the functional integrity of the mitochondria. At this field strength, about half of the internalized plasmid was found in the inner membrane or mitochondrial matrix, as determined by immunoelectron microscopy and Southern blot analysis of electroporated mitochondria treated with digitonin. We estimate that on average one plasmid molecule/mitochondrion reaches the matrix or inner membrane.
Animals↗
Laryngeal and oropharyngeal cancer, and alcohol dehydrogenase 3 and glutathione S-transferase M1 polymorphisms.
In this study the GSTmu phenotype and ADH genotype at the ADH3 locus were investigated in a group of 39 alcoholic men with upper respiratory/digestive tract cancer: 21 with oropharyngeal cancer and 18 with laryngeal cancer. The results are compared with those of a control group of 37 alcoholic men without alcohol-related medical complications. Of the control subjects, 48% were found to be GSTmu deficient [GSTmu(-)] and 19% carried the ADH(3)1/ADH(3)1 genotype. In the laryngeal cancer patients, a significantly elevated frequency of both the GSTmu(-) (78%) and ADH(3)1/ADH(3)1 genotype (56%) was observed, relative to the control group. On the basis of this result, the risk of laryngeal cancer associated with the GSTmu(-) and ADH(3)1/ADH(3)1 genotypic combination within the population of alcoholics was estimated to be 12.9 with a 95% confidence interval of 1.8-92 (P < 0.01) relative to alcoholic individuals who have GSTmu [GSTmu(+)] and are not ADH(3)1/ADH(3)1. Thus, alcoholics who are GSTmu(-) and ADH(3)1/ADH(3)1 have at least an 80% greater risk of developing laryngeal cancer than alcoholics who are GSTmu(+) and who are not ADH(3)1/ADH(3)1. In addition, the oropharyngeal cancer patients had excess frequencies of both GSTmu(-) (62%) and ADH(3)1/ADH(3)1 (43%) relative to the control group, but these excess frequencies were not statistically significant. The GSTmu(-) and ADH(3)1/ADH(3)1 genotypic combination may be a constitutional risk factor for laryngeal cancer among alcoholics.
Adult↗