Showing papers by "Transgene SA published in 2001"

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism.

Abstract: Targeting of adenovirus (Ad)-encoded therapeutic genes to specific cell types has become a major goal in gene therapy. Redirecting the specificity of infection requires the abrogation of the natural interaction between the viral fiber and its cellular receptors (CAR) and the simultaneous introduction of a new binding specificity into the viral capsid. To abrogate the natural affinity of the fiber, we have mutated residues presumed to be directly or indirectly involved in CAR-binding in the knob domain of the fiber protein. These residues are located in the AB loop (Ser408) and in the DG loop (Tyr491, Ala494, Ala503). The mutations Ser408Glu, Tyr491Asp, Ala494Asp and Ala503Asp did not prevent the incorporation of trimeric fibers in the viral capsid but led to loss of CAR binding in vitro. Infectivity of the mutant viruses could be restored in vitro by introducing a ligand at the C-terminal end of the knob, confirming that the reduced infectivity of the fiber-modified virus was due to an impaired interaction of the viral particle with the CAR receptor. However, after systemic delivery, the in vivo biodistribution of impaired CAR-binding viruses without addition of a specific ligand was not altered when compared with wild-type Ad.

Regulation of gap junctional communication by a pro-inflammatory cytokine in cystic fibrosis transmembrane conductance regulator-expressing but not cystic fibrosis airway cells

Abstract: Airway inflammation is orchestrated by cell-cell interactions involving soluble mediators and cell adhesion molecules. Alterations in the coordination of the multicellular process of inflammation may play a major role in the chronic lung disease state of cystic fibrosis (CF). The aim of this study was to determine whether direct cell-cell interactions via gap junctional communication is affected during the inflammatory response of the airway epithelium. We have examined the strength of intercellular communication and the activation of nuclear factor-κB (NF-κB) in normal (non-CF) and CF human airway cell lines stimulated with tumor necrosis factor-α (TNF-α). TNF-α induced maximal translocation of NF-κB into the nucleus of non-CF as well as CF airway cells within 20 minutes. In non-CF cells, TNF-α progressively decreased the extent of intercellular communication. In contrast, gap junctional communication between CF cells exposed to TNF-α remained unaltered. CF results from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Interestingly, transfer of wild-type CFTR into CF cells by adenovirus-mediated infection was associated with the recovery of TNF-α-induced uncoupling. These results suggest that expression of functional CFTR is necessary for regulation of gap junctional communication by TNF-α. Gap junction channels close during the inflammatory response, therefore limiting the intercellular diffusion of signaling molecules, and thereby the recruitment of neighboring cells. Defects in this mechanism may contribute to the excessive inflammatory response of CF airway epithelium.

Immunotherapy of established tumors in mice by intratumoral injection of an adenovirus vector harboring the human IL-2 cDNA: induction of CD8(+) T-cell immunity and NK activity.

Abstract: Intratumoral (i.t.) injections of an adenovirus encoding the human interleukin-2 (IL-2) under the control of the RSV (Ad-pRSV-IL-2) or CMV (Ad-pCMV-IL-2) promoter were performed in established mastocytoma P815 tumors in B6D2 mice. Both early and long-term survival were found increased in mice treated with Ad-pCMV-IL-2 as compared with those obtained with Ad-pRSV-IL-2: tumor regression was observed in 30--50% of mice for the former and 5--15% for the latter. Difference in efficacy between the two vectors was directly correlated to the amount of IL-2 produced i.t. between 24 and 48 hours postinjection, which reached 10--20 ng/tumor for Ad-pCMV-IL-2 and 0.3--0.5 ng/tumor for Ad-pRSV-IL-2. In both cases, expression in the tumor was clearly detectable for a period of 7--10 days postinjection. Serum IL-2 was not detectable in mice treated with Ad-pRSV-IL-2, whereas expression peaked at a total of 1--2 ng at 24 hours but declined very rapidly in the Ad-pCMV-IL-2-treated group. Constant production of IL-2 inside the tumor was necessary for successful therapy because i.t. injections of recombinant IL-2 at levels up to 1 microg for five consecutive days did not lead to antitumoral activity. Evidence of induced systemic immunity following Ad-pCMV-IL-2 injections was obtained from rechallenge experiments in which tumor-free mice after treatment rejected a subsequent contralateral injection of a lethal dose of P815 tumor cells and from the observation that regression of nontreated tumors occurred in animals bearing bilateral tumors that were treated i.t. in a single tumor with Ad-pCMV-IL-2. P815-specific cytotoxic T lymphocytes (CTL) were found specifically in spleen cells from cured mice or rechallenged mice but not in control mice. Interestingly, limiting dilution analysis of anti-P815 CTL precursor (CTLp) frequency revealed a significant increase in mice cured of their tumor as compared to that obtained in naive mice or control mice treated or not with Ad-IL-2 but whose tumor was growing. In vivo depletion of T-cell subsets, as well as natural killer cells at the time of i.t. injections with Ad-pCMV-IL-2, demonstrated that both CD8(+) T cells and natural killer cells, but not CD4(+) T cells, were required for successful therapy. Finally, mice preimmunized with Ad-null viruses were severely compromised in their capacity to eradicate established P815 tumors after Ad-pCMV-IL-2 therapy, at least when neutralizing antibody titers reached a critical level.

Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid.

Abstract: Background One of the main drawbacks of synthetic, non-viral gene vectors is their relatively low in vivo efficiency when compared with viral vectors. The present paper describes the use of a partially fluorinated glycerophosphoethanolamine (F-PE), a close analog of DOPE, which, as a helper lipid with the cationic lipopolyamine pcTG90, increases its in vitro and in vivo gene transfer capability to a larger extent than DOPE. Methods To evaluate the contribution of F-PE to lipoplex-mediated gene transfer, the effect of including F-PE in lipoplexes formulated with the lipopolyamine pcTG90 for various pcTG90/DOPE/F-PE molar ratios [1:(1−x):x; 1:(2−y):y] was examined. For the in vitro analyses on human lung carcinoma epithelial A549 cells, the lipoplexes were formulated with the luciferase reporter plasmid pTG11033 using various N/P ratios (from 10 to 0.8, N=number of pcTG90 amines, P=number of DNA phosphates). The in vivo analyses were performed (1) with the luciferase reporter plasmid pCMV-Luc, which gives higher luciferase expression in the lung than pcTG11033; (2) with pcTG90/co-lipid(s) (1:2) lipoplexes which yield higher expression than the (1:1) formulations; and (3) by intravenous (iv) injection into the tail vein of mice. Results The efficiency of the F-PE lipoplexes to transfect in vitro A549 cells was significantly higher (5–90-fold) than that of DOPE lipoplexes, when formulated in HEPES. However, when formulated in 5% glucose, both co-lipids display a comparable transfection helper potential. Most remarkably, an up to eight-fold increase of luciferase expression could be measured in the lung after iv injection of pcTG90/F-PE (1:2) N/P 5 lipoplexes as compared with the pcTG90/DOPE lipoplexes. It led also to higher luciferase expression than PEI(ExGen500)/pCMV-Luc N/P 10 polyplexes. Besides expression in lung, low levels of luciferase expression were also observed in heart, spleen and liver. Conclusion The present work, showing a higher in vitro and in vivo transfection potential for lipoplexes formulated with a partially fluorinated co-lipid as compared with its analogous DOPE lipoplexes or PEI polyplexes, indicates that ‘fluorinated’ lipoplexes are attractive candidates for in vivo applications. Copyright © 2001 John Wiley & Sons, Ltd.

Regression of primary hepatocarcinoma in cancer-prone transgenic mice by local interferon-γ delivery is associated with macrophages recruitment and nitric oxide production

Abstract: The clinical potential of tumor therapies must be evaluated using animal models closely resembling human cancers. We investigated the impact of locally delivered interferon-gamma (IFN-gamma) on primary hepatocarcinoma spontaneously developed by T-SV40 transgenic mice. A single intratumor injection of adenovirus IFN-gamma was sufficient enough to induce in vivo production of biologically active IFN-gamma, as assessed by STAT1 activation. IFN-gamma secretion led to the regression of primary tumor, principally by apoptosis of tumor hepatocytes. The lack of T-cells infiltrates in the liver upon treatment excluded a role of a specific immune response. In contrast, indirect pathways may include tumoricidal function of macrophages. Indeed, they were massively recruited in the entire liver under IFN-gamma treatment; transmigration through hepatic blood vessels could be observed and co-localization with damaged hepatocytes was obvious. This correlated with nonparenchymal liver cell iNOS expression and high level of NO in hepatic extracts. Moreover, in vitro experiments showed that NO releasing agents induced cell death of freshly isolated tumor hepatocytes, suggesting that NO could be one of the major effector molecules. Altogether, these observations defined an important role of IFN-gamma in controlling tumor development in a model of primary hepatocarcinoma.

On the possible role of muscle in the pathogenesis of spinal muscular atrophy

Abstract: Spinal muscular atrophy (SMA) is a common human inherited disease characterized by degeneration of motoneurons and muscular atrophy. SMA results from deletions or mutations of the SMN (survival motor neuron) gene. A nerve-muscle coculture model, consisting of human muscle cells innervated by rat embryonic spinal cord explants, was used to study the pathogenesis of SMA. Previous studies have shown that myotubes formed by fusion of satellite muscle cells from patients with SMA I or SMA II (but not SMA III) underwent a characteristic degeneration 1-3 weeks after innervation. To correlate this cellular study with a molecular approach, we used reverse transcriptase-polymerase chain reaction (RT-PCR), and showed that SMN mRNAs were expressed throughout the fusion of normal satellite muscle cells with two peaks, the first appearing prior to the onset of fusion and the second one or two days before innervation. When satellite muscle cells from patients with SMA I or II were used, only the first peak was observed. Because in these cases the SMN telomeric gene (SMNtel) is deleted, it was concluded that the contribution of SMNtel-dependent mRNAs to the second peak is predominant in normal myogenesis and involved in maturation of myotubes. In addition, diseased satellite muscle cells did not fuse at the same rate as normal satellite muscle cells. Studies on myf-5, a muscle specific transcription factor family, showed that its expression was impaired during the fusion of satellite muscle cells from patients with SMA I or II compared with normal satellite muscle cells. Taken together, these observations suggest that (a) there is a muscle specific expression pattern of SMN, and (b) SMN probably plays a crucial role in maintenance of a functional motor unit, by allowing muscle cells to correctly differentiate and to allow motoneuron survival.

Effects of first generation E1E3-deleted and second generation E1E3E4-deleted/modified adenovirus vectors on human endothelial cell death

Abstract: Adenoviral vectors are promising tools for pulmonary vascular gene transfer. In first generation vectors, the viral E4 region is preserved (E4+ Ad), but E4 is deleted in second generation vectors (E4- Ad). These vectors were compared for their toxicity in human endothelial cells in terms of apoptosis and necrosis. Infection with E4+ Ad vectors reduced whereas E4- Ad vectors enhanced apoptosis under normal culture conditions. Furthermore, E4+ Ad protected against apoptosis induced by growth factor deprivation, while E4- Ad enhanced apoptosis triggered by ceramide. Ad vectors containing different E4 open reading frames, alone or in different combinations, showed similar effects to E4- Ad, leaving the viral genes that might be responsible for reducing apoptosis unidentified at the present time. As previously observed with E4+ Ad devoid of transgene, E4+ Ad carrying beta-galactosidase or green fluorescent protein under the control of either the RSV or CMV promoter also reduced apoptosis triggered by growth factor deprivation. In contrast, E4+ Ad containing a CFTR expression cassette did not reduce apoptosis, and E4- Ad with CFFR showed increased toxicity. We conclude that Ad vectors may have important effects on the control of apoptosis in transfected cells, depending on the residual expression of viral genes. This effect can be complicated by the action of transgene expression on cell survival.

Combination product intended for carrying out a cytotoxic treatment, in particular an antitumour treatment, in a mammal

Abstract: The present invention relates to a combination product comprising at least one nucleic acid containing a sequence encoding a polypeptide or interest and at least one phospholipid of interest, for use which is simultaneous, consecutive or spread out over time, characterized in that said polypeptide and phospholipid of interest have cytotoxic activity.

Device for the administration of a composition in a conduit of a human or animal body

Abstract: The instrument incorporates metal, plastic or mineral perforators or cutters designed to make blind holes in the wall of a duct and a distributor for contacting a medicinal compound with the holes. The cutters or perforators can be extended radially relative to the axis of the instrument, e.g. by means of an inflatable chamber. The substance is distributed through a chamber with radial orifices.

Bases moléculaires de l'oncogenèse

Abstract: Les vingt-cinq dernieres annees ont permis des avancees spectaculaires dans le domaine de la recherche sur le cancer. L'ensemble de ces connaissances decrit le cancer comme une maladie faisant intervenir des changements dynamiques dans le genome cellulaire. La cancerogenese chez l'homme est un mecanisme en plusieurs etapes, dont chacune reflete des alterations genetiques aboutissant a la transformation progressive d'une cellule humaine en une cellule maligne. Durant cette transformation, les cellules tumorales detournent les circuits de regulation classiques qui gouvernent la proliferation cellulaire et l'homeostasie. Dans cette revue, nous decrirons les mecanismes moleculaires impliques dans la tumorigenese et leurs consequences.

Combination product for carrying out a cytotoxic treatment in a mammal

Abstract: The present invention relates to a combination product comprising at least one nucleic acid containing a sequence encoding a polypeptide or interest and at least one phospholipid of interest, for use which is simultaneous, consecutive or spread out over time, characterized in that said polypeptide and phospholipid of interest have cytotoxic activity.