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Dive into the research topics where Stéphanie Philippe is active.

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Featured researches published by Stéphanie Philippe.


Proceedings of the National Academy of Sciences of the United States of America | 2006

Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo

Stéphanie Philippe; Chamsy Sarkis; Martine Barkats; Hamid Mammeri; Charline Ladroue; Caroline Petit; Jacques Mallet; Che Serguera

Lentivirus-derived vectors are among the most promising viral vectors for gene therapy currently available, but their use in clinical practice is limited by the associated risk of insertional mutagenesis. We have overcome this problem by developing a nonintegrative lentiviral vector derived from HIV type 1 with a class 1 integrase (IN) mutation (replacement of the 262RRK motif by AAH). We generated and characterized HIV type 1 vectors carrying this deficient enzyme and expressing the GFP or neomycin phosphotransferase transgene (NEO) under control of the immediate early promoter of human CMV. These mutant vectors efficiently transduced dividing cell lines and nondividing neural primary cultures in vitro. After transduction, transient GFP fluorescence was observed in dividing cells, whereas long-term GFP fluorescence was observed in nondividing cells, consistent with the viral genome remaining episomal. Moreover, G418 selection of cells transduced with vectors expressing the NEO gene showed that residual integration activity was lower than that of the intact IN by a factor of 500–1,250. These nonintegrative vectors were also efficient in vivo, allowing GFP expression in mouse brain cells after the stereotactic injection of IN-deficient vector particles. Thus, we have developed a generation of lentiviral vectors with a nonintegrative phenotype of great potential value for secure viral gene transfer in clinical applications.


PLOS ONE | 2011

Retinal Degeneration Progression Changes Lentiviral Vector Cell Targeting in the Retina

Maritza Calame; M. Cachafeiro; Stéphanie Philippe; Karine Schouwey; M. Tekaya; D. Wanner; Chamsy Sarkis; Corinne Kostic; Yvan Arsenijevic

In normal mice, the lentiviral vector (LV) is very efficient to target the RPE cells, but transduces retinal neurons well only during development. In the present study, the tropism of LV has been investigated in the degenerating retina of mice, knowing that the retina structure changes during degeneration. We postulated that the viral transduction would be increased by the alteration of the outer limiting membrane (OLM). Two different LV pseudotypes were tested using the VSVG and the Mokola envelopes, as well as two animal models of retinal degeneration: light-damaged Balb-C and Rhodopsin knockout (Rho-/-) mice. After light damage, the OLM is altered and no significant increase of the number of transduced photoreceptors can be obtained with a LV-VSVG-Rhop-GFP vector. In the Rho-/- mice, an alteration of the OLM was also observed, but the possibility of transducing photoreceptors was decreased, probably by ongoing gliosis. The use of a ubiquitous promoter allows better photoreceptor transduction, suggesting that photoreceptor-specific promoter activity changes during late stages of photoreceptor degeneration. However, the number of targeted photoreceptors remains low. In contrast, LV pseudotyped with the Mokola envelope allows a wide dispersion of the vector into the retina (corresponding to the injection bleb) with preferential targeting of Müller cells, a situation which does not occur in the wild-type retina. Mokola-pseudotyped lentiviral vectors may serve to engineer these glial cells to deliver secreted therapeutic factors to a diseased area of the retina.


PLOS ONE | 2013

Rapid Cohort Generation and Analysis of Disease Spectrum of Large Animal Model of Cone Dystrophy

Corinne Kostic; Simon G. Lillico; Sylvain V. Crippa; Nicolas Grandchamp; Héloïse Pilet; Stéphanie Philippe; Zen Lu; Tim King; Jacques Mallet; Chamsy Sarkis; Yvan Arsenijevic; C. B. A. Whitelaw

Large animal models are an important resource for the understanding of human disease and for evaluating the applicability of new therapies to human patients. For many diseases, such as cone dystrophy, research effort is hampered by the lack of such models. Lentiviral transgenesis is a methodology broadly applicable to animals from many different species. When conjugated to the expression of a dominant mutant protein, this technology offers an attractive approach to generate new large animal models in a heterogeneous background. We adopted this strategy to mimic the phenotype diversity encounter in humans and generate a cohort of pigs for cone dystrophy by expressing a dominant mutant allele of the guanylate cyclase 2D (GUCY2D) gene. Sixty percent of the piglets were transgenic, with mutant GUCY2D mRNA detected in the retina of all animals tested. Functional impairment of vision was observed among the transgenic pigs at 3 months of age, with a follow-up at 1 year indicating a subsequent slower progression of phenotype. Abnormal retina morphology, notably among the cone photoreceptor cell population, was observed exclusively amongst the transgenic animals. Of particular note, these transgenic animals were characterized by a range in the severity of the phenotype, reflecting the human clinical situation. We demonstrate that a transgenic approach using lentiviral vectors offers a powerful tool for large animal model development. Not only is the efficiency of transgenesis higher than conventional transgenic methodology but this technique also produces a heterogeneous cohort of transgenic animals that mimics the genetic variation encountered in human patients.


Genetic Vaccines and Therapy | 2011

Influence of insulators on transgene expression from integrating and non-integrating lentiviral vectors

Nicolas Grandchamp; Dorothée Henriot; Stéphanie Philippe; Lahouari Amar; Suzanna Ursulet; Che Serguera; Jacques Mallet; Chamsy Sarkis

BackgroundThe efficacy and biosafety of lentiviral gene transfer is influenced by the design of the vector. To this end, properties of lentiviral vectors can be modified by using cis-acting elements such as the modification of the U3 region of the LTR, the incorporation of the central flap (cPPT-CTS) element, or post-transcriptional regulatory elements such as the woodchuck post-transcriptional regulatory element (WPRE). Recently, several studies evaluated the influence of the incorporation of insulators into the integrating lentiviral vector genome on transgene expression level and position effects.MethodsIn the present study, the influence of the matrix attachment region (MAR) of the mouse immunoglobulin-κ (Ig-κ) or the chicken lysozyme (ChL) gene was studied on three types of HIV-1-derived lentiviral vectors: self-inactivating (SIN) lentiviral vectors (LV), double-copy lentiviral vectors (DC) and non-integrating lentiviral vectors (NILVs) in different cell types: HeLa, HEK293T, NIH-3T3, Raji, and T Jurkat cell lines and primary neural progenitors.Results and DiscussionOur results demonstrate that the Ig-κ MAR in the context of LV slightly increases transduction efficiency only in Hela, NIH-3T3 and Jurkat cells. In the context of double-copy lentiviral vectors, the Ig-κ MAR has no effect or even negatively influences transduction efficiency. In the same way, in the context of non-integrating lentiviral vectors, the Ig-κ MAR has no effect or even negatively influences transduction efficiency, except in differentiated primary neural progenitor cells.The ChL MAR in the context of integrating and non-integrating lentiviral vectors shows no effect or a decrease of transgene expression in all tested conditions.ConclusionsThis study demonstrates that MAR sequences not necessarily increase transgene expression and that the effect of these sequences is probably context dependent and/or vector dependent. Thus, this study highlights the importance to consider a MAR sequence in a given context. Moreover, other recent reports pointed out the potential effects of random integration of insulators on the expression level of endogenous genes. Taken together, these results show that the use of an insulator in a vector for gene therapy must be well assessed in the particular therapeutic context that it will be used for, and must be balanced with its potential genotoxic effects.


Molecular Therapy | 2005

92. Efficient Trangene Expression in Central Nervous System through a Non-Integrative Lentiviral Vector

Stéphanie Philippe; Che Serguera; Sébastien Bonnel; Christelle Vêtu; Martine Barkats; Caroline Petit; Marc Abitbol; Chamsy Sarkis; Jacques Mallet

Lentiviral vectors are widely used in experimental gene transfer and are a promising tool for clinical applications, particularly for the treatment of neurodegenerative diseases. However, one of the most important limits to the application of this technology to humans is the risk of insertional mutagenesis through the integration of the viral genome into the host cell chromatin. This risk remains poorly evaluated and uncontrolled. In order to eliminate this risk, we have developed a lentiviral vector derived from the human immunodeficiency virus type 1 lacking integration property. After cell transduction, the vector genome persists in an extrachromosomic circular form in the nucleus and allows an effective and stable transgene expression in vitro and in vivo.


Human Gene Therapy | 2012

Lentiviral-directed transgenesis in swine is an efficient tool to study human dominant genetic diseases

Corinne Kostic; Nicolas Grandchamp; Tim King; Sylvain V. Crippa; Stéphanie Philippe; Simon G. Lillico; Chamsy Sarkis; Jacques Mallet; Yvan Arsenijevic; Bruce Whitelaw


Archive | 2012

TRANSIENT EXPRESSION VECTORS, PREPARATION AND USES THEREOF

Chamsy Sarkis; Dorothée Altemir; Nicolas Grandchamp; Stéphanie Philippe


Investigative Ophthalmology & Visual Science | 2012

Analysis Of Photoreceptor Abnormality In Gucy2d E837D/R838S Transgenic Pigs

Corinne Kostic; Tim King; Crippa Sylvain; Stéphanie Philippe; Simon G. Lillico; Chamsy Sarkis; Jacques Mallet; Yvan Arsenijevic; Bruce Whitelaw


Investigative Ophthalmology & Visual Science | 2010

Lentiviral Vector Tropism In Degenerating Retinas

M. Calame; M. Tekaya; A. Maillard; M. Cachafeiro; Stéphanie Philippe; Chamsy Sarkis; Jacques Mallet; Corinne Kostic; Yvan Arsenijevic


Investigative Ophthalmology & Visual Science | 2010

Rpe65-Gene Transfer Using an Integration-Deficient Lentiviral Vector

Corinne Kostic; Stéphanie Philippe; Sylvain V. Crippa; Marijana Samardzija; Vérène Pignat; D. Wanner; C. Grimm; Chamsy Sarkis; Jacques Mallet; Yvan Arsenijevic

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Chamsy Sarkis

Centre national de la recherche scientifique

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Tim King

University of Edinburgh

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D. Wanner

University of Lausanne

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M. Tekaya

University of Lausanne

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