Charles Coutelle

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.

In utero gene therapy: the case for.

adopted a UNESCO developed statement that would significantly prohibit human germline experimentation. Without approval, scientists or others engaging in this type of experimentation should face severe professional sanctions in addition to civil and criminal penalties. Patients and research subjects consent to treatment or research participation after being informed of the risks and benefits. Parents or guardians give permission for similar activities involving children, although there is usually a higher standard of safety and benefit. Although similar permission might be extended to lateterm fetuses with even more rigorous cost/benefit standards, this model’s extension to all of gestation and to generations of those yet to be conceived is not plausible. The public has an appropriate role in such decisionmaking. It would be a poor public policy decision if full patienthood was conferred on the more than 85% of conceptuses and fetuses that do not survive the normal gestation period or experience early neonatal death. Treatment of generally nonviable fetuses, even with powerful genetic methods and parental permission, should be avoided. Making young fetuses into patients only serves to confuse compassion with eugenics.

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.

Genetic analysis of DNA from single human oocytes: a model for preimplantation diagnosis of cystic fibrosis.

Gene sequences in human oocytes were studied to investigate the possibility of diagnosing inherited or sporadic genetic disease before implantation after in vitro fertilisation. By specific amplification the possibility of analysing the DNA from single human oocytes for a specific gene was shown, and genotypes for markers closely linked to cystic fibrosis and Duchenne muscular dystrophy were determined. Single oocytes were used to approximate the total amount of DNA present in a single cell taken for biopsy from a 4-16 cell blastocyst. With a new technique for specific DNA amplification, the polymerase chain reaction, these data can be obtained within several hours of cell isolation. Extreme care must be taken to avoid any contamination of the sample with DNA from other sources. With this technique genotyping for single gene disorders is feasible with an accuracy and on a time scale that would allow implantation of the zygote after in vitro fertilisation without freezing.

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.

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 β-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 (AdRSVβgal) into the amniotic cavity of foetal mice between 13 to 16 days post coitus, β-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.

Characterisation of LMD virus-like nanoparticles self-assembled from cationic liposomes, adenovirus core peptide μ (mu) and plasmid DNA

Liposome:mu:DNA (LMD) is a ternary nucleic acid delivery system built around the μ (mu) peptide associated with the condensed core complex of the adenovirus. LMD is prepared by precondensing plasmid DNA (D) with mu peptide (M) in a 1:0.6 (w/w) ratio and then combining these mu:DNA (MD) complexes with extruded cationic liposomes (L) resulting in a final lipid:mu:DNA ratio of 12:0.6:1 (w/w/w). Correct buffer conditions, reagent concentrations and rates of mixing are all crucial to success. However, once optimal conditions are established, homogeneous LMD particles (120 ± 30 nm) will result that each appear to comprise an MD particle encapsulated within a cationic bilammellar liposome. LMD particles can be formulated reproducibly, they are amenable to long-term storage (>1 month) at −80°C and are stable to aggregation at a plasmid DNA concentration up to 5 mg/ml (15 mM nucleotide concentration). Furthermore, LMD transfections are significantly more time and dose efficient in vitro than cationic liposome-plasmid DNA (LD) transfections. Transfection times as short as 10 min and plasmid DNA doses as low as 0.001 μg/well result in significant gene expression. LMD transfections will also take place in the presence of biological fluids (eg up to 100% serum) giving 15–25% the level of gene expression observed in the absence of serum. Results from confocal microscopy experiments using fluorescent-labelled LMD particles suggest that endocytosis is not a significant barrier to LMD transfection, although the nuclear membrane still is. We also confirm that topical lung transfection in vivo by LMD is at least equal in absolute terms with transfection mediated by GL-67:DOPE:DMPE-PEG5000 (1:2:0.05 m/m/m), an accepted ‘gold-standard’ non-viral vector system for topical lung transfection, and is in fact at least six-fold more dose efficient. All these features make LMD an important new non-viral vector platform system from which to derive tailor-made non-viral delivery systems by a process of systematic modular upgrading.

Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector.

An ideal gene therapy vector should enable persistent transgene expression without limitations of safety and reproducibility. Here we report the development of a non-viral episomal plasmid DNA (pDNA) vector that appears to fulfil these criteria. This pDNA vector combines a scaffold/matrix attachment region (S/MAR) with a human liver-specific promoter (α1-antitrypsin (AAT)) in such a way that long-term expression is enabled in murine liver following hydrodynamic injection. Long-term expression is demonstrated by monitoring the longitudinal luciferase expression profile for up to 6 months by means of in situ bioluminescent imaging. All relevant control pDNA constructs expressing luciferase are unable to sustain significant transgene expression beyond 1 week post-administration. We establish that this shutdown of expression is due to promoter methylation. In contrast, the S/MAR element appears to inhibit methylation of the AAT promoter thereby preventing transgene silencing. Although this vector appears to be maintained as an episome throughout, we have no evidence for its establishment as a replicating entity. We conclude that the combination of a mammalian, tissue-specific promoter with the S/MAR element is sufficient to drive long-term episomal pDNA expression of genes in vivo.

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.

Successful expression of β-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 β-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 β-galactosidase encoding vector, was found in almost all fetal and neonatal organs and in the maternal liver. Expression of the β-galactosidase transgene was detected in many fetal tissues by RT-PCR. High β-galactosidase production was shown by immuno-histochemistry predominantly in the liver, where about 30% 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 80% 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.