Nicolas Grandchamp

This article provides new data on the leishmania cycle and could help in the development of new therapeutic strategies against the disease, in particular in the design of effective vaccines.

This review provides vaccination state-of-the-art and discusses the new generations of vaccines based on nucleic acids, their recent progresses as their issues and challenges.

This review provides a human gene therapy state-of-the-art and discusses the recent marketing approvals as well as their issues and challenges.

HIV-1 derived vectors are among the most efficient for gene transduction in mammalian tissues. As the parent virus, they carry out vector genome insertion into the host cell chromatin. Consequently, their preferential integration in transcribed genes raises several conceptual and safety issues. To address part of these questions, HIV-derived vectors have been engineered to be nonintegrating. This was mainly achieved by mutating HIV-1 integrase at functional hotspots of the enzyme enabling the… Voir plus

HIV-1 derived vectors are among the most efficient for gene transduction in mammalian tissues. As the parent virus, they carry out vector genome insertion into the host cell chromatin. Consequently, their preferential integration in transcribed genes raises several conceptual and safety issues. To address part of these questions, HIV-derived vectors have been engineered to be nonintegrating. This was mainly achieved by mutating HIV-1 integrase at functional hotspots of the enzyme enabling the development of streamlined nuclear DNA circles functional for transgene expression. Few integrase mutant vectors have been successfully tested so far for gene transfer. They are cleared with time in mitotic cells, but stable within nondividing retina cells or neurons. Here, we compared six HIV vectors carrying different integrases, either wild type or with different mutations (D64V, D167H, Q168A, K186Q+Q214L+Q216L, and RRK262-264AAH) shown to modify integrase enzymatic activity, oligomerization, or interaction with key cellular cofactor of HIV DNA integration as LEDGF/p75 or TNPO3. We show that these mutations differently affect the transduction efficiency as well as rates and patterns of integration of HIV-derived vectors suggesting their different processing in the nucleus. Surprisingly and most interestingly, we report that an integrase carrying the D167H substitution improves vector transduction efficiency and integration in both HEK-293T and primary CD34+ cells. Voir moins

Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In… Voir plus

Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable. Voir moins

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… Voir plus

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. Voir moins

Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide… Voir plus

Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects. Voir moins

he 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… Voir plus

he 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.
In 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.
This 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. Voir moins

The techniques of gene transfer allow transitory or permanent genetic modification of cells for therapy or genetic engineering. Viral vectors derived from lentiviruses, developed over the past fifteen years, are revolutionizing the field by their ease of production and high efficiency to transduce most cell types.
In the laboratory, we developed Non-Integrating Lentiviral Vectors (NILVs) by introducing a mutation in the catalytic domain of the viral integrase. These vectors constitute a… Voir plus

The techniques of gene transfer allow transitory or permanent genetic modification of cells for therapy or genetic engineering. Viral vectors derived from lentiviruses, developed over the past fifteen years, are revolutionizing the field by their ease of production and high efficiency to transduce most cell types.
In the laboratory, we developed Non-Integrating Lentiviral Vectors (NILVs) by introducing a mutation in the catalytic domain of the viral integrase. These vectors constitute a platform of choice for a specific integration system. We thus tested their ability to vectorize the specific integrase of PhiC31 phage and its substrate. The natural function of this recombinase is to catalyze recombination between the AttB and AttP sites. We have developed two types of NILVs, one that bears the recombinase and the other containing the sequence of interest accompanied by the AttB site. In theory, in a cell that is cotransduced with these NILVs, the PhiC31 recombinase should catalyze recombination between the AttB site contained in the NILV and and a pseudo AttP site (pAttP, homologous to the AttP site), present in the genome of eukaryotic cells. This event would result in the specific integration of NILVs substrate in a pseudo site.
The work presented here can be divided in two parts. The first one consists in improving the performance of the recombinase and the NILVs. The second part describes the vectorization of PhiC31 system in NILVs in various settings in vitro and in vivo.
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