Abderrahim Mahfoudi - Academia.edu (original) (raw)

Papers by Abderrahim Mahfoudi

Cancer Research, 2020

Background: VISTA is a B7 family protein described as a negative checkpoint of T cell responses, ... more Background: VISTA is a B7 family protein described as a negative checkpoint of T cell responses, both in autoimmunity and cancer murine models. K01401-020 is a novel IND enabling anti-VISTA antibody. In vitro, when incubated with human blood cells, K01401-020 stimulates NK cells, monocytes and cytokine production, contributing to T cell activation. In vivo, in non-human primates, K01401-020 notably induced dendritic cell activation (Loukili et al, AACR 2019). However, so far, no in vivo demonstration of anti-VISTA pharmacodynamic effect on T cells has been demonstrated beyond mouse models. Thus, we explored the effect of anti-VISTA antibody on CD8 T cell specific activation in non-human primates, using SIV immunization as a model. Methods: The detection of T cell response amplification by immune-checkpoint inhibitors is very challenging in primates during standard PK or safety studies in the absence of adequate antigen stimulation. KLH is described as an immunogen in monkeys but ind...

Molecular Endocrinology, 1995

Molecular Therapy, 2000

Numerous diseases are linked to the absence or insufficient concentration of a specific plasma pr... more Numerous diseases are linked to the absence or insufficient concentration of a specific plasma protein. Gene transfer is an appealing strategy for correction of such diseases. We report high and sustained plasma secretion of human secreted alkaline phosphatase and of human Factor IX by skeletal muscle of mice. This was obtained by delivering square-wave unipolar electric pulses of low field strength (200 V/cm) and long duration (20 ms) to skeletal muscle previously injected with plasmid DNA encoding for the secreted protein. This intramuscular electrotransfer method allows 30- to 150-fold increase in reporter protein secretion, compared to simple plasmid DNA injection. This increase allows one to obtain values of up to 2200 ng/ml of a reporter circulating protein. Moreover, this high level of secretion remains stable for several months.

Molecular Therapy, 2001

Intramuscular plasmid DNA injection results in long-term but low and variable expression of the i... more Intramuscular plasmid DNA injection results in long-term but low and variable expression of the injected genes. Optimization is difficult because the mechanism of naked DNA uptake by the cells in vivo is not yet determined. Here we used injections of plasmid DNA encoding luciferase to further characterize this mechanism. We analyzed the kinetics of naked DNA uptake by means of DNase I or heparin injections, using the level of luciferase expression as the indicator of DNA uptake. We demonstrated that in vivo heparin inhibits DNA uptake without affecting the expression of DNA internalized by means of electric pulses. Inhibition by heparin is dose dependent and compatible with the competition for the binding to a receptor. As shown also with DNase I, DNA uptake by muscle cells is slow: a progressive accumulation of the DNA in the myofibers can be found for at least 4 hours after naked DNA injection. Physical presence of DNA molecules during the uptake period, but not later, was confirmed by the facilitation of DNA uptake with appropriate electric pulses. Therefore, uptake proceeds for the entire time during which intact DNA is present in the extracellular compartment. Our results support evidence for a DNA uptake mechanism based on receptor-mediated endocytosis.

The Journal of Steroid Biochemistry and Molecular Biology, 1993

Peroxisome proliferator activated receptors are ligand activated transcription factors belonging ... more Peroxisome proliferator activated receptors are ligand activated transcription factors belonging to the nuclear hormone receptor superfamily. Three cDNAs encoding such receptors have been isolated from Xenopus laevis (xPPAR alpha, beta, and gamma). Furthermore, the gene coding for xPPAR beta has been cloned, thus being the first member of this subfamily whose genomic organization has been solved. Functionally, xPPAR alpha as well as its mouse and rat homologs are thought to play an important role in lipid metabolism due to their ability to activate transcription of a reporter gene through the promoter of the acyl-CoA oxidase (ACO) gene. ACO catalyzes the rate limiting step in the peroxisomal beta-oxidation of fatty acids. Activation is achieved by the binding of xPPAR alpha on a regulatory element (DR1) found in the promoter region of this gene, xPPAR beta and gamma are also able to recognize the same type of element and are, as PPAR alpha, able to form heterodimers with retinoid X receptor. All three xPPARs appear to be activated by synthetic peroxisome proliferators as well as by naturally occurring fatty acids, suggesting that a common mode of action exists for all the members of this subfamily of nuclear hormone receptors.

Current Cardiology Reports, 2000

Proceedings of the National Academy of Sciences, 1993

The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, be... more The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, beta, and gamma) regulate the peroxisomal beta-oxidation of fatty acids by induction of the acyl-CoA oxidase gene that encodes the rate-limiting enzyme of the pathway. Gel retardation and cotransfection assays revealed that PPAR alpha heterodimerizes with retinoid X receptor beta (RXR beta; RXR is the receptor for 9-cis-retinoic acid) and that the two receptors cooperate for the activation of the acyl-CoA oxidase gene promoter. The strongest stimulation of this promoter was obtained when both receptors were exposed simultaneously to their cognate activators. Furthermore, we show that natural fatty acids, and especially polyunsaturated fatty acids, activate PPARs as potently as does the hypolipidemic drug Wy 14,643, the most effective activator known so far. Moreover, we discovered that the synthetic arachidonic acid analogue 5,8,11,14-eicosatetraynoic acid is 100 times more effective than ...

Proceedings of the National Academy of Sciences, 1995

The estrogen receptor (ER) stimulates transcription of target genes by means of its two transcrip... more The estrogen receptor (ER) stimulates transcription of target genes by means of its two transcriptional activation domains, AF-1 in the N-terminal part of the receptor and AF-2 in its ligand-binding domain. AF-2 activity is dependent upon a putative amphipathic alpha-helix between residues 538 and 552 in the mouse ER. Point mutagenesis of conserved hydrophobic residues within this region reduces estrogen-dependent transcriptional activation without affecting hormone and DNA binding significantly. Here we show that these mutations dramatically alter the pharmacology of estrogen antagonists. Both tamoxifen and ICI 164,384 behave as strong agonists in HeLa cells expressing the ER mutants. In contrast to the wild-type ER, the mutant receptors maintain nuclear localization and DNA-binding activity after ICI 164,384 treatment. Structural alterations in AF-2 caused by gene mutations such as those described herein or by estrogen-independent signaling pathways may account for the insensitivi...

Molecular Therapy, 2002

Efficient cell electrotransfection can be achieved using combinations of high-voltage (HV; 800 V/... more Efficient cell electrotransfection can be achieved using combinations of high-voltage (HV; 800 V/cm, 100 s) and low-voltage (LV; 80 V/cm, 100 ms) pulses. We have developed equipment allowing the generation of various HV and LV combinations with precise control of the lag between the HV and LV pulses. We injected luciferase-encoding DNA in skeletal muscle, before or after pulse delivery, and measured luciferase expression after various pulse combinations. In parallel, we determined permeabilization levels using uptake of 51 Cr-labeled EDTA. High voltage alone resulted in a high level of muscle permeabilization for 300 seconds, but very low DNA transfer. Combinations of one HV pulse followed by one or four LV pulses did not prolong the high permeabilization level, but resulted in a large increase in DNA transfer for lags up to 100 seconds in the case of one HV + one LV and up to 3000 seconds in the case of one HV + four LV. DNA expression also reached similar levels when we injected the DNA between the HV and LV pulses. We conclude that the role of the HV pulse is limited to muscle cell permeabilization and that the LV pulses have a direct effect on DNA. In vivo DNA electrotransfer is thus a multistep process that includes DNA distribution, muscle permeabilization, and DNA electrophoresis.

Molecular Therapy, 2002

We have developed a new gene regulation system for gene therapy. This system consists of two expr... more We have developed a new gene regulation system for gene therapy. This system consists of two expression cassettes; one expresses the human peroxisome proliferator-activated receptor ␥ (PPAR␥), and the other expresses the therapeutic gene under the control of multiple peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) linked to a basal promoter. Using direct injection of plasmid DNA into skeletal muscle or myocardium of rodents and oral administration of clinically approved PPAR␥ activators, we demonstrate that reporter gene expression can be induced more than 25-fold. We show that oral administration of PPAR␥ activator at intervals separated by several months results in repeated pulses of high-level reporter gene expression. We also document a PPAR␥ activator dose-response effect on reporter gene expression. This is the first report of a gene regulation system that makes use of a human transcription factor and that may be safer than chimeric transcription factors for human gene therapy.

Molecular and Cellular Endocrinology, 1994

A procedure to culture Xenopus laeuis hepatocytes that allows the cells in primary culture to be ... more A procedure to culture Xenopus laeuis hepatocytes that allows the cells in primary culture to be subjected to gene transfer experiments has been developed. The cultured cells continue to present tissue-specific markers such as expression of the albumin gene or estrogen-controlled vitellogenin gene expression, which are both restricted to liver. Two efficient and reproducible gene transfer procedures have been adapted to the Xenopus hepatocytes, namely lipofection and calcium phosphate-mediated precipitation. The transcription of transfected reporter genes controlled by estrogen-, glucocorticoid-or peroxisome proliferator-response elements was stimulated by endogenous or co-transfected receptor in a ligand-dependent manner. Furthermore, the expression of a reporter gene under the control of the entire promoter of the vitellogenin Bl gene mimicked the expression of the chromosomal vitellogenin gene with respect to basal and estrogen-induced activity. Thus, this culture-transfection system will prove very useful to study the regulation of genes expressed in the liver under the control of various hormones or xenobiotics.

The Journal of Gene Medicine, 2003

BackgroundIn vivo gene transfer to skeletal muscle is a promising strategy for the treatment of m... more BackgroundIn vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscular disorders and for the systemic delivery of therapeutic proteins. Nevertheless, for a safe and effective protein production, the spatial and temporal control of gene expression is critical. The existing regulating systems rely on the use of an exogenously regulatory protein and/or an inducer drug whose pharmacological properties are of major concerns for therapeutic applications in humans. Therefore, new strategies based on endogenous regulatable elements have been explored.MethodsGene expression profiles of skeletal muscle submitted or not to electrical pulses and harvested at different times were compared using the Affymetrix GeneChip technology. The endogenous metallothionein promoter was studied by Northern blot and semiquantitative and quantitative RT‐PCR. The inducibility of the metallothionein I promoter placed in a plasmid exogenous context was studied using the murine SEA...

Journal of Biological Chemistry, 1995

Human Gene Therapy, 2002

Pharmacologic gene regulation is a key technology, necessary to achieve safe, long-term gene tran... more Pharmacologic gene regulation is a key technology, necessary to achieve safe, long-term gene transfer. The approaches described in the scientific literature all share in common the creation of artificial transcription factors by fusing a DNA-binding domain, a drug-binding domain and a transcription activation domain. These transcription factors activate the transgene expression upon binding of the pharmacologic agent (antibiotics of the tetracycline family, insect hormone, progesterone antagonist, or immunosuppressor drug) to the drug-binding domain. The major limitations to the use of these systems for human gene and cell therapies are the toxicity of the inducer molecule and the immunogenicity of the chimeric transcription factor. Thus, the gene regulation systems should operate with clinically approved drugs with safety records that do not conflict with the therapeutic gene expression regimen. This work focuses on the characterization of the immunogenicity of a tetracycline-activated transcription factor commonly used in preclinical gene therapy, rtTA2-M2, and its impact on reporter gene expression. We demonstrate that intramuscular injection of plasmid or adenoviral vectors encoding rtTA-M2 in outbred primates generates a cellular and humoral immune response to this transcription factor. The immune response to rtTA2-M2 blunts the duration of the expression the rtTA2-M2-controlled transgene in primates, presumably by destruction of the cells that coexpress rtTA2-M2 and the reporter or therapeutic gene. This immune response may result directly from the vectors used in this study, which prompts the development of new gene transfer vectors enabling safe and efficient pharmacologic gene regulation in clinic.

Gene, 2001

The improvement of gene therapy vectors would benefit from the availability of a reporter gene th... more The improvement of gene therapy vectors would benefit from the availability of a reporter gene that can be used for long-term studies in immunocompetent laboratory animals. We describe the construction and characterization of a novel reporter gene, murine secreted embryonic alkaline phosphatase (MUSEAP). We demonstrate by gene transfer in skeletal muscle of immunocompetent mice that MUSEAP is efficiently secreted and detected in the bloodstream and that injection of an increasing dose of DNA leads to a dose-dependent increase of plasma MUSEAP activity. We also show that the expression of MUSEAP under the control of a constitutive promoter is stable for 1 year and that the activity of MUSEAP in the bloodstream reflects the changes in the transcription rate of its gene. These properties make MUSEAP the only reporter gene that can be used for somatic gene transfer into immunocompetent mice in order to study the impact of gene transfer vectors of metabolic, developmental or environmental factors on long-term gene expression.

Endocrinology, 1992

Immunohistochemistry with a polyclonal antibody raised against human plasma fibronectin (Fn) was ... more Immunohistochemistry with a polyclonal antibody raised against human plasma fibronectin (Fn) was used to determine the localization of Fn in endometrial sections of guinea pig uteri isolated at the first, fourth, sixth, or tenth day of the estrous cycle. Immunoreactive Fn was constantly visualized in the endometrial stroma but absent from the epithelial layer. Fn was detected in the uterine lumen on the first or fourth day of the estrous cycle and was absent from the other sections. To determine the origin of this luminal Fn the ability of subcultured endometrial cells to produce Fn was tested, and the hormonal regulation of Fn secretion was studied. Cells were treated by estradiol alone or in association with progesterone, progesterone alone, or untreated. Whatever the hormonal treatment, stromal cells constantly secreted immunoreactive Fn into the culture medium. In the same way, the amount of Fn synthesized and basally secreted by epithelial cells was not affected by any hormonal treatments. However, Fn was found in the apical secretions of the untreated or estradiol-treated epithelial cells but was undetectable in the apical compartment when the epithelial cells were treated by progesterone alone or in association with estradiol. These results indicate that Fn is constitutively secreted by stromal cells and that subcultured epithelial cells of guinea pig endometrium secrete Fn from both their basal and apical membrane domains. However, the apical secretion of Fn is specifically suppressed by progesterone.

Biology of the Cell, 1992

Summary— Stromal and glandular epithelial (GE) cells were isolated from guinea‐pig endometrium an... more Summary— Stromal and glandular epithelial (GE) cells were isolated from guinea‐pig endometrium and grown to near confluency (6–8 days) in primary culture on plastic surfaces in a serum‐supplemented medium (SSM). The stromal cells were subcultured on plastic dishes and maintained for 72 h in SSM. Then SSM was replaced by a chemically defined medium (CDM) and the stromal cells grown to confluency (5–7 days). The GE cells were subcultured in CDM, on a basement membrane matrix (Matrigel) applied to permeable Millicell‐PC filters, and grown to confluency (5 days). Homogeneity of the subcultured endometrial cell populations was ascertained immunocytochemically. The filter‐cultured GE monolayers were polarized morphologically, and displayed epithelialspecific specialized structures. These monolayers had functional tight junctions as verified by a measurable transepithelial resistance. The subcultured cell populations were distinguished by an analysis of their cellular and secretory protein...

Biology of the Cell, 1993

Summary— Peroxisome proliferators regulate the transcription of genes by activating ligand‐depend... more Summary— Peroxisome proliferators regulate the transcription of genes by activating ligand‐dependent transcription factors, which, due to their structure and function, can be assigned to the superfamily of nuclear hormone receptors. Three such peroxisome proliferator‐activated receptors (PPARα, β, and γ) have been cloned in Xenopus laevis. Their mRNAs are expressed differentially; xPPARα and β but not xPPARγ are expressed in oocytes and embryos. In the adult, expression of xPPARα and β appears to be ubiquitous, and xPPARγ is mainly observed in adipose tissue and kidney. Immunocytochemical analysis revealed that PPARs are nuclear proteins, and that their cytoplasmic‐nuclear translocation is independent of exogenous activators. A target gene of PPARs is the gene encoding acyl‐CoA oxidase (ACO), which catalyzes the rate‐limiting step in the peroxisomal β‐oxidation of fatty acids. A peroxisome proliferator response element (PPRE), to which PPARs bind, has been identified within the prom...

Biology of the Cell, 1991

Epithelial glands were isolated from guinea-pig endometrium. In order to reduce the requirement f... more Epithelial glands were isolated from guinea-pig endometrium. In order to reduce the requirement for a serum supplement and the contamination by non epithelial cells in primary culture, various coatings of the culture dishes were tested using serum-free Ham's F12 containing defined chemicals including 17 beta-estradiol. While epithelial glands seeded on culture dishes coated with Matrigel, a basement membrane matrix-failed to spread, they formed on poly-D-lysine plus serum-coated dishes, a subconfluent monolayer (5-7 days) enriched in cytokeratin-immunostained cells (78%). Cells from subconfluent primary cultures, obtained on poly-D-lysine plus serum-coated dishes in serum-free hormonally defined medium, were passaged on Matrigel-coated dishes in serum-free hormonally defined medium. These subcultures contained, at confluence (4-5 days), a high percentage (greater than 95%) of cytokeratin-immunostained cells. These monolayers consisted of well-differentiated cells which exhibited ultrastructural features characteristic of endometrial epithelial cells. Moreover, these confluent cells contained 50% immunostained nuclei for progesterone receptors. Progesterone receptor amounts decreased in confluent subcultures treated with progesterone and became undetectable after long-term treatment, suggesting responsiveness of these cells to progesterone. This culture system provides a well-defined model for the study of protein synthesis and secretion by endometrial glandular epithelial cells under hormonal control.

Cancer Research, 2020

Background: VISTA is a B7 family protein described as a negative checkpoint of T cell responses, ... more Background: VISTA is a B7 family protein described as a negative checkpoint of T cell responses, both in autoimmunity and cancer murine models. K01401-020 is a novel IND enabling anti-VISTA antibody. In vitro, when incubated with human blood cells, K01401-020 stimulates NK cells, monocytes and cytokine production, contributing to T cell activation. In vivo, in non-human primates, K01401-020 notably induced dendritic cell activation (Loukili et al, AACR 2019). However, so far, no in vivo demonstration of anti-VISTA pharmacodynamic effect on T cells has been demonstrated beyond mouse models. Thus, we explored the effect of anti-VISTA antibody on CD8 T cell specific activation in non-human primates, using SIV immunization as a model. Methods: The detection of T cell response amplification by immune-checkpoint inhibitors is very challenging in primates during standard PK or safety studies in the absence of adequate antigen stimulation. KLH is described as an immunogen in monkeys but ind...

Molecular Endocrinology, 1995

Molecular Therapy, 2000

Numerous diseases are linked to the absence or insufficient concentration of a specific plasma pr... more Numerous diseases are linked to the absence or insufficient concentration of a specific plasma protein. Gene transfer is an appealing strategy for correction of such diseases. We report high and sustained plasma secretion of human secreted alkaline phosphatase and of human Factor IX by skeletal muscle of mice. This was obtained by delivering square-wave unipolar electric pulses of low field strength (200 V/cm) and long duration (20 ms) to skeletal muscle previously injected with plasmid DNA encoding for the secreted protein. This intramuscular electrotransfer method allows 30- to 150-fold increase in reporter protein secretion, compared to simple plasmid DNA injection. This increase allows one to obtain values of up to 2200 ng/ml of a reporter circulating protein. Moreover, this high level of secretion remains stable for several months.

Molecular Therapy, 2001

Intramuscular plasmid DNA injection results in long-term but low and variable expression of the i... more Intramuscular plasmid DNA injection results in long-term but low and variable expression of the injected genes. Optimization is difficult because the mechanism of naked DNA uptake by the cells in vivo is not yet determined. Here we used injections of plasmid DNA encoding luciferase to further characterize this mechanism. We analyzed the kinetics of naked DNA uptake by means of DNase I or heparin injections, using the level of luciferase expression as the indicator of DNA uptake. We demonstrated that in vivo heparin inhibits DNA uptake without affecting the expression of DNA internalized by means of electric pulses. Inhibition by heparin is dose dependent and compatible with the competition for the binding to a receptor. As shown also with DNase I, DNA uptake by muscle cells is slow: a progressive accumulation of the DNA in the myofibers can be found for at least 4 hours after naked DNA injection. Physical presence of DNA molecules during the uptake period, but not later, was confirmed by the facilitation of DNA uptake with appropriate electric pulses. Therefore, uptake proceeds for the entire time during which intact DNA is present in the extracellular compartment. Our results support evidence for a DNA uptake mechanism based on receptor-mediated endocytosis.

The Journal of Steroid Biochemistry and Molecular Biology, 1993

Peroxisome proliferator activated receptors are ligand activated transcription factors belonging ... more Peroxisome proliferator activated receptors are ligand activated transcription factors belonging to the nuclear hormone receptor superfamily. Three cDNAs encoding such receptors have been isolated from Xenopus laevis (xPPAR alpha, beta, and gamma). Furthermore, the gene coding for xPPAR beta has been cloned, thus being the first member of this subfamily whose genomic organization has been solved. Functionally, xPPAR alpha as well as its mouse and rat homologs are thought to play an important role in lipid metabolism due to their ability to activate transcription of a reporter gene through the promoter of the acyl-CoA oxidase (ACO) gene. ACO catalyzes the rate limiting step in the peroxisomal beta-oxidation of fatty acids. Activation is achieved by the binding of xPPAR alpha on a regulatory element (DR1) found in the promoter region of this gene, xPPAR beta and gamma are also able to recognize the same type of element and are, as PPAR alpha, able to form heterodimers with retinoid X receptor. All three xPPARs appear to be activated by synthetic peroxisome proliferators as well as by naturally occurring fatty acids, suggesting that a common mode of action exists for all the members of this subfamily of nuclear hormone receptors.

Current Cardiology Reports, 2000

Proceedings of the National Academy of Sciences, 1993

The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, be... more The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, beta, and gamma) regulate the peroxisomal beta-oxidation of fatty acids by induction of the acyl-CoA oxidase gene that encodes the rate-limiting enzyme of the pathway. Gel retardation and cotransfection assays revealed that PPAR alpha heterodimerizes with retinoid X receptor beta (RXR beta; RXR is the receptor for 9-cis-retinoic acid) and that the two receptors cooperate for the activation of the acyl-CoA oxidase gene promoter. The strongest stimulation of this promoter was obtained when both receptors were exposed simultaneously to their cognate activators. Furthermore, we show that natural fatty acids, and especially polyunsaturated fatty acids, activate PPARs as potently as does the hypolipidemic drug Wy 14,643, the most effective activator known so far. Moreover, we discovered that the synthetic arachidonic acid analogue 5,8,11,14-eicosatetraynoic acid is 100 times more effective than ...

Proceedings of the National Academy of Sciences, 1995

The estrogen receptor (ER) stimulates transcription of target genes by means of its two transcrip... more The estrogen receptor (ER) stimulates transcription of target genes by means of its two transcriptional activation domains, AF-1 in the N-terminal part of the receptor and AF-2 in its ligand-binding domain. AF-2 activity is dependent upon a putative amphipathic alpha-helix between residues 538 and 552 in the mouse ER. Point mutagenesis of conserved hydrophobic residues within this region reduces estrogen-dependent transcriptional activation without affecting hormone and DNA binding significantly. Here we show that these mutations dramatically alter the pharmacology of estrogen antagonists. Both tamoxifen and ICI 164,384 behave as strong agonists in HeLa cells expressing the ER mutants. In contrast to the wild-type ER, the mutant receptors maintain nuclear localization and DNA-binding activity after ICI 164,384 treatment. Structural alterations in AF-2 caused by gene mutations such as those described herein or by estrogen-independent signaling pathways may account for the insensitivi...

Molecular Therapy, 2002

Efficient cell electrotransfection can be achieved using combinations of high-voltage (HV; 800 V/... more Efficient cell electrotransfection can be achieved using combinations of high-voltage (HV; 800 V/cm, 100 s) and low-voltage (LV; 80 V/cm, 100 ms) pulses. We have developed equipment allowing the generation of various HV and LV combinations with precise control of the lag between the HV and LV pulses. We injected luciferase-encoding DNA in skeletal muscle, before or after pulse delivery, and measured luciferase expression after various pulse combinations. In parallel, we determined permeabilization levels using uptake of 51 Cr-labeled EDTA. High voltage alone resulted in a high level of muscle permeabilization for 300 seconds, but very low DNA transfer. Combinations of one HV pulse followed by one or four LV pulses did not prolong the high permeabilization level, but resulted in a large increase in DNA transfer for lags up to 100 seconds in the case of one HV + one LV and up to 3000 seconds in the case of one HV + four LV. DNA expression also reached similar levels when we injected the DNA between the HV and LV pulses. We conclude that the role of the HV pulse is limited to muscle cell permeabilization and that the LV pulses have a direct effect on DNA. In vivo DNA electrotransfer is thus a multistep process that includes DNA distribution, muscle permeabilization, and DNA electrophoresis.

Molecular Therapy, 2002

We have developed a new gene regulation system for gene therapy. This system consists of two expr... more We have developed a new gene regulation system for gene therapy. This system consists of two expression cassettes; one expresses the human peroxisome proliferator-activated receptor ␥ (PPAR␥), and the other expresses the therapeutic gene under the control of multiple peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) linked to a basal promoter. Using direct injection of plasmid DNA into skeletal muscle or myocardium of rodents and oral administration of clinically approved PPAR␥ activators, we demonstrate that reporter gene expression can be induced more than 25-fold. We show that oral administration of PPAR␥ activator at intervals separated by several months results in repeated pulses of high-level reporter gene expression. We also document a PPAR␥ activator dose-response effect on reporter gene expression. This is the first report of a gene regulation system that makes use of a human transcription factor and that may be safer than chimeric transcription factors for human gene therapy.

Molecular and Cellular Endocrinology, 1994

A procedure to culture Xenopus laeuis hepatocytes that allows the cells in primary culture to be ... more A procedure to culture Xenopus laeuis hepatocytes that allows the cells in primary culture to be subjected to gene transfer experiments has been developed. The cultured cells continue to present tissue-specific markers such as expression of the albumin gene or estrogen-controlled vitellogenin gene expression, which are both restricted to liver. Two efficient and reproducible gene transfer procedures have been adapted to the Xenopus hepatocytes, namely lipofection and calcium phosphate-mediated precipitation. The transcription of transfected reporter genes controlled by estrogen-, glucocorticoid-or peroxisome proliferator-response elements was stimulated by endogenous or co-transfected receptor in a ligand-dependent manner. Furthermore, the expression of a reporter gene under the control of the entire promoter of the vitellogenin Bl gene mimicked the expression of the chromosomal vitellogenin gene with respect to basal and estrogen-induced activity. Thus, this culture-transfection system will prove very useful to study the regulation of genes expressed in the liver under the control of various hormones or xenobiotics.

The Journal of Gene Medicine, 2003

BackgroundIn vivo gene transfer to skeletal muscle is a promising strategy for the treatment of m... more BackgroundIn vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscular disorders and for the systemic delivery of therapeutic proteins. Nevertheless, for a safe and effective protein production, the spatial and temporal control of gene expression is critical. The existing regulating systems rely on the use of an exogenously regulatory protein and/or an inducer drug whose pharmacological properties are of major concerns for therapeutic applications in humans. Therefore, new strategies based on endogenous regulatable elements have been explored.MethodsGene expression profiles of skeletal muscle submitted or not to electrical pulses and harvested at different times were compared using the Affymetrix GeneChip technology. The endogenous metallothionein promoter was studied by Northern blot and semiquantitative and quantitative RT‐PCR. The inducibility of the metallothionein I promoter placed in a plasmid exogenous context was studied using the murine SEA...

Journal of Biological Chemistry, 1995

Human Gene Therapy, 2002

Pharmacologic gene regulation is a key technology, necessary to achieve safe, long-term gene tran... more Pharmacologic gene regulation is a key technology, necessary to achieve safe, long-term gene transfer. The approaches described in the scientific literature all share in common the creation of artificial transcription factors by fusing a DNA-binding domain, a drug-binding domain and a transcription activation domain. These transcription factors activate the transgene expression upon binding of the pharmacologic agent (antibiotics of the tetracycline family, insect hormone, progesterone antagonist, or immunosuppressor drug) to the drug-binding domain. The major limitations to the use of these systems for human gene and cell therapies are the toxicity of the inducer molecule and the immunogenicity of the chimeric transcription factor. Thus, the gene regulation systems should operate with clinically approved drugs with safety records that do not conflict with the therapeutic gene expression regimen. This work focuses on the characterization of the immunogenicity of a tetracycline-activated transcription factor commonly used in preclinical gene therapy, rtTA2-M2, and its impact on reporter gene expression. We demonstrate that intramuscular injection of plasmid or adenoviral vectors encoding rtTA-M2 in outbred primates generates a cellular and humoral immune response to this transcription factor. The immune response to rtTA2-M2 blunts the duration of the expression the rtTA2-M2-controlled transgene in primates, presumably by destruction of the cells that coexpress rtTA2-M2 and the reporter or therapeutic gene. This immune response may result directly from the vectors used in this study, which prompts the development of new gene transfer vectors enabling safe and efficient pharmacologic gene regulation in clinic.

Gene, 2001

The improvement of gene therapy vectors would benefit from the availability of a reporter gene th... more The improvement of gene therapy vectors would benefit from the availability of a reporter gene that can be used for long-term studies in immunocompetent laboratory animals. We describe the construction and characterization of a novel reporter gene, murine secreted embryonic alkaline phosphatase (MUSEAP). We demonstrate by gene transfer in skeletal muscle of immunocompetent mice that MUSEAP is efficiently secreted and detected in the bloodstream and that injection of an increasing dose of DNA leads to a dose-dependent increase of plasma MUSEAP activity. We also show that the expression of MUSEAP under the control of a constitutive promoter is stable for 1 year and that the activity of MUSEAP in the bloodstream reflects the changes in the transcription rate of its gene. These properties make MUSEAP the only reporter gene that can be used for somatic gene transfer into immunocompetent mice in order to study the impact of gene transfer vectors of metabolic, developmental or environmental factors on long-term gene expression.

Endocrinology, 1992

Immunohistochemistry with a polyclonal antibody raised against human plasma fibronectin (Fn) was ... more Immunohistochemistry with a polyclonal antibody raised against human plasma fibronectin (Fn) was used to determine the localization of Fn in endometrial sections of guinea pig uteri isolated at the first, fourth, sixth, or tenth day of the estrous cycle. Immunoreactive Fn was constantly visualized in the endometrial stroma but absent from the epithelial layer. Fn was detected in the uterine lumen on the first or fourth day of the estrous cycle and was absent from the other sections. To determine the origin of this luminal Fn the ability of subcultured endometrial cells to produce Fn was tested, and the hormonal regulation of Fn secretion was studied. Cells were treated by estradiol alone or in association with progesterone, progesterone alone, or untreated. Whatever the hormonal treatment, stromal cells constantly secreted immunoreactive Fn into the culture medium. In the same way, the amount of Fn synthesized and basally secreted by epithelial cells was not affected by any hormonal treatments. However, Fn was found in the apical secretions of the untreated or estradiol-treated epithelial cells but was undetectable in the apical compartment when the epithelial cells were treated by progesterone alone or in association with estradiol. These results indicate that Fn is constitutively secreted by stromal cells and that subcultured epithelial cells of guinea pig endometrium secrete Fn from both their basal and apical membrane domains. However, the apical secretion of Fn is specifically suppressed by progesterone.

Biology of the Cell, 1992

Summary— Stromal and glandular epithelial (GE) cells were isolated from guinea‐pig endometrium an... more Summary— Stromal and glandular epithelial (GE) cells were isolated from guinea‐pig endometrium and grown to near confluency (6–8 days) in primary culture on plastic surfaces in a serum‐supplemented medium (SSM). The stromal cells were subcultured on plastic dishes and maintained for 72 h in SSM. Then SSM was replaced by a chemically defined medium (CDM) and the stromal cells grown to confluency (5–7 days). The GE cells were subcultured in CDM, on a basement membrane matrix (Matrigel) applied to permeable Millicell‐PC filters, and grown to confluency (5 days). Homogeneity of the subcultured endometrial cell populations was ascertained immunocytochemically. The filter‐cultured GE monolayers were polarized morphologically, and displayed epithelialspecific specialized structures. These monolayers had functional tight junctions as verified by a measurable transepithelial resistance. The subcultured cell populations were distinguished by an analysis of their cellular and secretory protein...

Biology of the Cell, 1993

Summary— Peroxisome proliferators regulate the transcription of genes by activating ligand‐depend... more Summary— Peroxisome proliferators regulate the transcription of genes by activating ligand‐dependent transcription factors, which, due to their structure and function, can be assigned to the superfamily of nuclear hormone receptors. Three such peroxisome proliferator‐activated receptors (PPARα, β, and γ) have been cloned in Xenopus laevis. Their mRNAs are expressed differentially; xPPARα and β but not xPPARγ are expressed in oocytes and embryos. In the adult, expression of xPPARα and β appears to be ubiquitous, and xPPARγ is mainly observed in adipose tissue and kidney. Immunocytochemical analysis revealed that PPARs are nuclear proteins, and that their cytoplasmic‐nuclear translocation is independent of exogenous activators. A target gene of PPARs is the gene encoding acyl‐CoA oxidase (ACO), which catalyzes the rate‐limiting step in the peroxisomal β‐oxidation of fatty acids. A peroxisome proliferator response element (PPRE), to which PPARs bind, has been identified within the prom...

Biology of the Cell, 1991

Epithelial glands were isolated from guinea-pig endometrium. In order to reduce the requirement f... more Epithelial glands were isolated from guinea-pig endometrium. In order to reduce the requirement for a serum supplement and the contamination by non epithelial cells in primary culture, various coatings of the culture dishes were tested using serum-free Ham's F12 containing defined chemicals including 17 beta-estradiol. While epithelial glands seeded on culture dishes coated with Matrigel, a basement membrane matrix-failed to spread, they formed on poly-D-lysine plus serum-coated dishes, a subconfluent monolayer (5-7 days) enriched in cytokeratin-immunostained cells (78%). Cells from subconfluent primary cultures, obtained on poly-D-lysine plus serum-coated dishes in serum-free hormonally defined medium, were passaged on Matrigel-coated dishes in serum-free hormonally defined medium. These subcultures contained, at confluence (4-5 days), a high percentage (greater than 95%) of cytokeratin-immunostained cells. These monolayers consisted of well-differentiated cells which exhibited ultrastructural features characteristic of endometrial epithelial cells. Moreover, these confluent cells contained 50% immunostained nuclei for progesterone receptors. Progesterone receptor amounts decreased in confluent subcultures treated with progesterone and became undetectable after long-term treatment, suggesting responsiveness of these cells to progesterone. This culture system provides a well-defined model for the study of protein synthesis and secretion by endometrial glandular epithelial cells under hormonal control.