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N Déglon

Publications and source records attributed to N Déglon.

32 records · Page 2Linked to original sources
PubMed2000

Efficient gene transfer and expression of biologically active glial cell line-derived neurotrophic factor in rat motoneurons transduced wit lentiviral vectors.

Several studies have shown the ability of human immunodeficiency virus type 1 (HIV1)-based lentiviral vectors to infect nondividing brain and retinal neurons with high efficiency and long-term expression of the transduced gene. We show that purified embryonic motoneurons can be efficiently (>95%) transduced in culture using an HIV1-based lentiviral vector encoding LacZ. Expression of beta-galactosidase was observed for at least 9 days in these conditions. Furthermore, motoneurons transduced with a lentiviral vector expressing glial cell line-derived neurotrophic factor survived in the absence of additional trophic support, showing that the overexpressed protein was biologically active. Our results demonstrate the potential of lentiviral vectors in studying the biological effects of proteins expressed in motoneurons and in the development of future gene therapy for motoneuron diseases.

Animals↗
PubMed1999

Lentiviral gene transfer to the nonhuman primate brain.

Lentiviral vectors infect quiescent cells and allow for the delivery of genes to discrete brain regions. The present study assessed whether stable lentiviral gene transduction can be achieved in the monkey nigrostriatal system. Three young adult Rhesus monkeys received injections of a lentiviral vector encoding for the marker gene beta galatosidase (beta Gal). On one side of the brain, each monkey received multiple lentivirus injections into the caudate and putamen. On the opposite side, each animal received a single injection aimed at the substantia nigra. The first two monkeys were sacrificed 1 month postinjection, while the third monkey was sacrificed 3 months postinjection. Robust incorporation of the beta Gal gene was seen in the striatum of all three monkeys. Stereological counts revealed that 930,218; 1,192,359; and 1,501,217 cells in the striatum were beta Gal positive in monkeys 1 (n = 2) and 3 (n = 1) months later, respectively. Only the third monkey had an injection placed directly into the substantia nigra and 187,308 beta Gal-positive cells were identified in this animal. The injections induced only minor perivascular cuffing and there was no apparent inflammatory response resulting from the lentivirus injections. Double label experiments revealed that between 80 and 87% of the beta Gal-positive cells were neurons. These data indicate that robust transduction of striatal and nigral cells can occur in the nonhuman primate brain for up to 3 months. Studies are now ongoing testing the ability of lentivirus encoding for dopaminergic trophic factors to augment the nigrostriatal system in nonhuman primate models of Parkinson's disease.

Animals↗
PubMed1998

Continuous delivery of human and mouse erythropoietin in mice by genetically engineered polymer encapsulated myoblasts.

The transplantation of polymer encapsulated myoblasts genetically engineered to secrete erythropoietin (Epo) may obviate the need for repeated parenteral administration of recombinant Epo as a treatment for chronic renal failure, cancer or AIDS-associated anemia. To explore this possibility, the human and mouse Epo cDNAs under the control of the housekeeping mouse PGK-1 promoter were transfected into mouse C2C12 myoblasts, which can be terminally differentiated upon exposure to low serum-containing media. Pools releasing 150 IU human Epo per 10(6) cells per day and 390 IU mouse Epo per 10(6) cells per day were selected. Polyether-sulfone (PES) capsules loaded with approximately 200,000 transfected myoblasts from these pools were implanted on the dorsal flank of DBA/2J, C3H and C57BL/6 mice. With human Epo secreting capsules, only a transient increase in the hematocrit occurred in DBA/2J mice, whereas no significant response was detected in C3H or C57BL/6 mice. On the contrary, all mice implanted with capsules releasing mouse Epo increased their hematocrit over 85% as early as 7 days after implantation and sustained these levels for at least 80 days. All retrieved implants released Epo and contained well preserved myoblasts. Moreover most capsules were surrounded by a neovascularization. Mice transplanted with nonencapsulated C2C12 cells releasing mouse Epo showed only a transitory elevation of their hematocrit reflecting the poor engraftment of injected myoblasts. These results indicate that polymer encapsulation of genetically engineered myoblasts is a promising approach for the long-term delivery of bioactive molecules, allowing the resolution of the shortcomings of free myoblast transfer.

Acquired Immunodeficiency Syndrome↗
PubMed1997

A gene therapy approach to regulated delivery of erythropoietin as a function of oxygen tension.

Current therapy for several forms of anemia involves a weekly regime of multiple subcutaneous injections of recombinant human erythropoietin (hEpo). In an effort to provide a physiologically regulated administration of erythropoietin, we are developing cell lines genetically engineered to release hEpo as a function of oxygen tension. C2C12 cells were transfected using a vector containing the hEpo cDNA driven by the hypoxia-responsive promoter to the murine phosphoglycerate kinase gene. In vitro, these cells showed a threefold increase in hEpo secretion as oxygen levels were shifted from 21% to 1.3% oxygen. To test in vivo response, C2C12-hEpo cells were encapsulated in a microporous membrane and implanted subcutaneously on the dorsal flank of DBA/2J mice. On average, serum hEpo levels in animals exposed to 7% oxygen were two-fold higher than values seen in their control counterparts kept at 21% oxygen. Similar studies employing rats confirmed that hEpo delivery is regulated as a function of oxygen tension. These results suggest the feasibility of developing an oxygen-regulated, encapsulated cell-based system for hEpo delivery.

Anemia↗
PubMed1996

Central nervous system delivery of recombinant ciliary neurotrophic factor by polymer encapsulated differentiated C2C12 myoblasts.

Neurotrophic factors hold promise for the treatment of neurodegenerative diseases. Intrathecal transplantation of polymer encapsulated cell lines genetically engineered to release neurotrophic factors provides a means to deliver them continuously behind the blood-brain barrier. Long-term delivery, however, may benefit from the use of conditionally mitotic cells to avoid the overgrowth observed with continuously dividing cell lines. Myoblast lines have all the advantages of dividing cell lines, i.e., unlimited availability, possibility for in vitro screening for the presence of pathogens, suitability for stable gene transfer and clonal selection. Furthermore they can be differentiated into a nonmitotic stage upon exposure to low-serum-containing medium. In this study, mouse C2C12 myoblasts were transfected with a pNUT expression vector containing the human ciliary neurotrophic factor (CNTF) gene. hCNTF expression and bioactivity were demonstrated by Northern blot, ELISA assay, and measurement of choline acetyltransferase (ChAT) activity in embryonic spinal cord motor neuron cultures. One C2C12 clone was found to secrete 200 ng of CNTF/10(6) cells per day. The rate of secretion of hCNTF was not altered upon differentiation of C2C12 myoblasts. A bromodeoxyuridine (BrdU) proliferation assay indicated that approximately 12% of the myoblasts continue to divide after 4 days in low-serum-containing medium. The presence of the herpes simplex thymidine kinase gene (HSV-tk) in the expression vector, however, provides a way to eliminate these dividing myoblasts upon exposure to ganciclovir, therefore increasing the safety of the encapsulation technology using established cell lines. Encapsulated hCNTF-C2C12 cells can partially rescue motor neurons from axotomy-induced cell death. In adult rats, intrathecal implantation of encapsulated hCNTF-C2C12 cells or control C2C12 confirmed the long-term survival of these cells and their potential use as a source of neurotophic factors for the treatment of neurodegenerative diseases.

Animals↗
PubMed1996

Intrathecal delivery of CNTF using encapsulated genetically modified xenogeneic cells in amyotrophic lateral sclerosis patients.

Neuronal growth factors hold promise for providing therapeutic benefits in various neurological disorders. As a means of ensuring adequate central nervous system delivery of growth factors and minimizing significant adverse side effects associated with systemic delivery methods, we have developed an ex vivo gene therapy approach for protein delivery using encapsulated genetically modified xenogeneic cells. Ciliary neurotrophic factor (CNTF) has been shown in various rodent models to reduce the motor neuron cell death similar to that seen in amyotrophic lateral sclerosis (ALS). The initial trials focusing on the systemic administration of CNTF for ALS have been discontinued as a result of major side effects, thus preventing determination of the potential efficacy of the molecule. In order to deliver CNTF directly to the nervous system, we conducted a phase I study in which six ALS patients were implanted with polymer capsules containing genetically engineered baby hamster kidney cells releasing approximately 0.5 microgram of human CNTF per day in vitro. The CNTF-releasing implants were surgically placed within the lumbar intrathecal space. Nanogram levels of CNTF were measured within the patients' cerebrospinal fluid (CSF) for at least 17 weeks post-transplantation, whereas it was undetectable before implantation. Intrathecal delivery of CNTF was not associated with the limiting side effects observed with systemic delivery. These results demonstrate that neurotrophic factors can be continuously delivered within the CSF of humans by an ex vivo gene therapy approach, opening new avenues for the treatment of neurological diseases.

Amyotrophic Lateral Sclerosis↗
PubMed1995

Fatty acids regulate Thy-1 antigen mRNA stability in T lymphocyte precursors.

In this study, we report the effect of fatty acids on the Thy-1 antigen mRNA decay. Low serum and synthetic medium culture conditions were used to demonstrate that fatty acids, which are important metabolites involved as second messengers in signal transduction, also influence the steady-state mRNA level. Detailed analysis demonstrated that polyunsaturated lipids attached to bovine serum albumin, such as linoleic, linolenic, and arachidonic acids, modulate gene expression specifically in the S1A T lymphoma cell line by inducing a 3-5-fold increase in the steady-state Thy-1 mRNA level, concomitant with a twofold increase in cell surface expression. A similar modulation was observed in the immature CD4-CD8- T cell precursors but not in mature thymocytes. Nuclear run-on and transfection experiments indicated that the observed Thy-1 mRNA level is post-transcriptionally regulated and that the presence of the coding region is sufficient for this adaptive response. A mechanism without a requirement for protein kinase C activation, but involving Ca2+ entry, could account for this difference in Thy-1 mRNA stability.

Animals↗
PubMed1992

Membrane mu poly(A) signal and 3' flanking sequences function as a transcription terminator for immunoglobulin-encoding genes.

Developmentally regulated mechanisms involving alternative RNA splicing and/or polyadenylation, as well as transcription termination, are implicated in controlling the levels of secreted mu (mu s), membrane mu (mu m) and delta immunoglobulin (Ig) heavy chain mRNAs during B cell differentiation (mu gene encodes the mu heavy chain). Using expression vectors constructed with genomic DNA segments composed of the mu m polyadenylation signal region, we analyzed poly(A) site utilization and termination of transcription in stably transfected myeloma cells and in murine fibroblast L cells. We found that the gene segment containing the mu m poly(A) signals, along with 536 bp of downstream flanking sequence, acted as a transcription terminator in both myeloma cells and L cell fibroblasts. Neither a 141-bp DNA fragment (which directed efficient polyadenylation at the mu m site), nor the 536-bp flanking nucleotide sequence alone, were sufficient to obtain a similar regulation. This shows that the mu m poly(A) region plays a central role in controlling developmentally regulated transcription termination by blocking downstream delta gene expression. Because this gene segment exhibited the same RNA processing and termination activities in fibroblasts, it appears that these processes are not tissue-specific.

Alternative Splicing↗
PubMed1991

Isolation from mouse fibroblasts of a cDNA encoding a new form of the fibroblast growth factor receptor (flg).

Structural definition of the receptors for neurotropic and angiogenic modulators such as fibroblast growth factors and related polypeptides will yield insight into the mechanisms that control early development, embryogenesis, organogenesis, wound repair and neovessel formation. We isolated 3 murine cDNAs encoding different binding domains of these receptors (flg). Comparison of these ectoplasmic portions showed that two of the forms corresponded to previously described murine molecules whereas the third one had a different ectoplasmic portion generated by specific changes in two regions. Interestingly, expression of this third form seems to be restricted in its tissue distribution. Such modifications could influence the ligand specificity of the different receptors and/or their binding affinity.

Amino Acid Sequence↗
PubMed1991

Translocation of the yeast dolichol-phosphate-mannose synthase into microsomal membranes.

Dolichol-phosphate-mannose synthase catalyzes the formation of Dolichol-phosphate-mannose from Dolichol-phosphate and GDP-mannose. Analysis of the primary amino acid sequence of the yeast enzyme predicts a luminal orientation of the enzyme in the endoplasmic reticulum. We analysed the translocation of the Dolichol-phosphate-mannose synthase into dog pancreatic microsomal membranes: resistance to proteolytic attack provides evidence of its luminal orientation and asks for a reevaluation of the topology of the reaction.

Animals↗
PubMedDate not supplied

Rescue of motoneurons from axotomy-induced cell death by polymer encapsulated cells genetically engineered to release CNTF.

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) results from the progressive loss of motoneurons, leading to death in a few years. Ciliary neurotrophic factor (CNTF), which decreases naturally occurring and axotomy-induced cell death, may result in slowing of motoneuron loss and has been evaluated as a treatment for ALS. Effective administration of this protein to motoneurons may be hampered by the exceedingly short half-life of CNTF, and the inability to deliver effective concentration into the central nervous system after systemic administration in vivo. The constitutive release of CNTF from genetically engineered cells may represent a solution to this delivery problem. In this work, baby hamster kidney (BHK) cells stably tranfected with a chimeric plasmid construct containing the gene for human or mouse CNTF were encapsulated in polymer fibers, which prevents immune rejection and allow long-term survival of the transplanted cells. In vitro bioassays show that the encapsulated transfected cells release bioactive CNTF. In vivo, systemic delivery of human and mouse CNTF from encapsulated cells was observed to rescue 26 and 27% more facial motoneurons, respectively, as compared to capsules containing parent BHK cells 1 wk postaxotomy in neonatal rats. With local application of CNTF on the nerve stump and by systemic delivery through repeated subcutaneous injections, 15 and 13% more rescue effects were observed. These data illustrate the potential of using encapsulated genetically engineered cells to continuously release CNTF to slow down motoneuron degeneration following axotomy and suggest that encapsulated cell delivery of neurotrophic factors may provide a general method for effective administration of therapeutic proteins for the treatment of neurodegenerative diseases.

Amyotrophic Lateral Sclerosis↗