Gene therapy: relevance and limits of vectors

The spectacular developments achieved in biology and biotechnology over the past few decades have completely modified biomedical research and opened up unprecedented therapeutic possibilities. In addition to the now classical genomics/post-genomics methods, these advances include the phenomenon of RNA interference, gene network building, ex vivo and in vivo gene transfer, and the construction of human disease models using various animal species. The treatment of obesity is a convincing example of such possibilities and of the power of gene transfer methods. Numerous diseases can benefit from gene therapy, particularly neurodegenerative diseases (brain and eye), currently lacking satisfactory approaches. Many proofs of principle have been established and human trials have been successful. However, gene therapy protocols still rely predominantly on virus-based vectors. Although they have been “disarmed”, these vectors still present risks for the patient, particularly of recombination, which should be carefully analyzed. Taking such risks into account and conducting appropriate research are the only way gene therapy will become routine treatment and achieve its remarkable therapeutic potential.Les développements spectaculaires de la biologie et de la biotechnologie, durant ces dernières décennies, ont bouleversé le paysage de la recherche biomédicale et offrent des possibilités thérapeutiques sans précédent. À coté des méthodes désormais classiques de la génomique/post-génomique, citons le phénomène d'ARN interférence, l'établissement de réseaux gènes/protéines, le transfert de gènes ex vivo et in vivo et la construction de modèles de maladies humaines chez différentes espèces animales. Un exemple emprunté au domaine de l'obésité illustre avec force ces nouvelles possibilités et la puissance des approches de transfert de gène. De nombreuses maladies peuvent bénéficier de la thérapie génique, en particulier les maladies neurodégénératives (cerveau et œil), pour lesquelles il n'existe pratiquement pas, à l'heure actuelle, d'alternatives satisfaisantes. De nombreuses preuves de principe ont été établies et des essais chez l'homme ont été couronnés de succès. Toutefois, la majorité des protocoles de thérapie génique font appel à des vecteurs dérivés de virus. Bien que « désarmés », ces vecteurs présentent encore des risques pour le patient, en particulier de recombinaison, qu'il est impératif d'analyser et de prendre en compte. Seuls cette prise de conscience et le développement de recherches appropriées permettront l'utilisation clinique, en routine, des outils de la thérapie génique et ainsi d'exploiter pleinement leur remarquable potentiel thérapeutique

Smoking-induced long-lasting modifications of human platelet serotonin catabolism through a MAO epigenetic regulation

Postulating that serotonin, secreted from smoking-activated platelets, could be involved in smoking-induced vascular modifications, we studied 115 men distributed in smokers (S), former smokers (FS) and never smokers (NS). The platelet serotonin content was similar in S and NS but lower in FS. This was unexpected because the monoamine oxidase (MAO) activity, which catabolizes serotonin, was inhibited during smoking. However, the amount of platelet MAO was higher in S and FS than in NS. The persistent elevated MAO amount in FS prompted us to study the methylation of its gene promoter in an additional series of patients: it was markedly lower for S and FS vs. NS due to cigarette smoke-induced increase of nucleic acid demethylase activity. This smoking-induced demethylation of the MAO gene promoter, resulting in high MAO amount persisting long after quitting smoking, has cardiovascular consequences and could impact fields such as behavior, mental health, and cancer

Control of small inhibitory RNA levels and RNA interference by doxycycline induced activation of a minimal RNA polymerase III promoter

RNA interference (RNAi) mediated by expression of short hairpin RNAs (shRNAs) is a powerful tool for efficiently suppressing target genes. The approach allows studies of the function of individual genes and may also be applied to human therapy. However, in many instances regulation of RNAi by administration of a small inducer molecule will be required. To date, the development of appropriate regulatory systems has been hampered by the few possibilities for modification within RNA polymerase III promoters capable of driving efficient expression of shRNAs. We have developed an inducible minimal RNA polymerase III promoter that is activated by a novel recombinant transactivator in the presence of doxycycline (Dox). The recombinant transactivator and the engineered promoter together form a system permitting regulation of RNAi by Dox-induced expression of shRNAs. Regulated RNAi was mediated by one single lentiviral vector, blocked the expression of green fluorescent protein (GFP) in a GFP-expressing HEK 293T derived cell line and suppressed endogenous p53 in wild-type HEK 293T, MCF-7 and A549 cells. RNA interference was induced in a dose- and time-dependent manner by administration of Dox, silenced the expression of both target genes by 90% and was in particular reversible after withdrawal of Dox

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

International audienceBACKGROUND: The 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. METHODS: 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. RESULTS AND DISCUSSION: Our 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. CONCLUSIONS: 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

Smoking Induces Long-Lasting Effects through a Monoamine-Oxidase Epigenetic Regulation

BACKGROUND: Postulating that serotonin (5-HT), released from smoking-activated platelets could be involved in smoking-induced vascular modifications, we studied its catabolism in a series of 115 men distributed as current smokers (S), never smokers (NS) and former smokers (FS) who had stopped smoking for a mean of 13 years. METHODOLOGY/PRINCIPAL FINDINGS: 5-HT, monoamine oxidase (MAO-B) activities and amounts were measured in platelets, and 5-hydroxyindolacetic acid (5-HIAA)--the 5-HT/MAO catabolite--in plasma samples. Both platelet 5-HT and plasma 5-HIAA levels were correlated with the 10-year cardiovascular Framingham relative risk (P<0.01), but these correlations became non-significant after adjustment for smoking status, underlining that the determining risk factor among those taken into account in the Framingham risk calculation was smoking. Surprisingly, the platelet 5-HT content was similar in S and NS but lower in FS with a parallel higher plasma level of 5-HIAA in FS. This was unforeseen since MAO-B activity was inhibited during smoking (P<0.00001). It was, however, consistent with a higher enzyme protein concentration found in S and FS than in NS (P<0.001). It thus appears that MAO inhibition during smoking was compensated by a higher synthesis. To investigate the persistent increase in MAO-B protein concentration, a study of the methylation of its gene promoter was undertaken in a small supplementary cohort of similar subjects. We found that the methylation frequency of the MAOB gene promoter was markedly lower (P<0.0001) for S and FS vs. NS due to cigarette smoke-induced increase of nucleic acid demethylase activity. CONCLUSIONS/SIGNIFICANCE: This is one of the first reports that smoking induces an epigenetic modification. A better understanding of the epigenome may help to further elucidate the physiopathology and the development of new therapeutic approaches to tobacco addiction. The results could have a larger impact than cardiovascular damage, considering that MAO-dependent 5-HT catabolism is also involved in addiction, predisposition to cancer, behaviour and mental health

A Novel and Efficient Gene Transfer Strategy Reduces Glial Reactivity and Improves Neuronal Survival and Axonal Growth In Vitro

Background: The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi). Methods and Findings: In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model -scratched primary cultured astrocytes- Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi. Conclusions: Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration after central nervous system lesions. Copyright: © 2009 Desclaux et al.This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Sante et de la Recherche Medicale (INSERM), the Universite Pierre et Marie Curie - Paris 6 (UPMC), the Universite de Montpellier 2, Verticale, Demain Debout, and the Institut de Recherche sur la Moelle epiniere et l’Encephale (IRME).Peer Reviewe

Hypersonic flows for reentry problems

INRIA and GAMNI/SMAI have organized a Workshop open to the international scientific community on hypersonic flows for reentry problems which was held in Palais des Congres d'Antibes in January 1990 (part I) and April 1991 (part II). This workshop was focussed on the issues of validation of numerical methodolies for the computation of high-Mach number flows, and gathered experts in Scientifical computing, fluid mechanics and experimentalists associated with the R and D Hermes program. Several volumes are devoted to the results and the detailed syntheses. This report presents a general synthesis of the motivations for this initiative, the development of the meetings, and the main conclusions drawn. A shorter version of this document has served as notes corresponding to a lecture given at an AGARD meeting

Obsessive compulsive disorder- prevalence in Xhosaspeaking schizophrenia patients

Obsessive compulsive disorder (OCD) has been reported in up to 31% of  schizophrenia sufferers. This study evaluated the presence of OCD in a  Xhosa-speaking schizophrenia group. Xhosa patients (N = 509, including 100  sibships) with schizophrenia were recruited from hospital and community settings. The patients underwent a structured clinical interview for the presence  of lifetime co-morbid schizo-phrenia and OCD. Only 3 patients (0.5%) fulfilled criteria for OCD. No concordance for OCD was noted in the  sibship group. Our findings differ from those in other parts of the world, and if replicated, might suggest unique protective environmental or genetic factors  for OCD in certain ethnic groups