Thérapie génique du système nerveux central: Considérations générales sur les vecteurs viraux pour le transfert de gène dans le cerveau

International audienceThe last decade have nourished strong doubts on the beneficial prospects of gene therapy for curing fatal diseases. However, this climate of reservations is currently being transcended by the publication of several successful clinical protocols, restoring confidence in the opportunity of therapeutic gene transfer. A strong sign of this present enthusiasm for gene therapy by clinicians and industrials is the market approval of the therapeutic viral vector Glybera, the first commercial product in Europe of this class of drug. This new field of medicine is particularly attractive when considering therapies for a number of neurological disorders, most of which are desperately waiting for a satisfactory treatment. The central nervous system is indeed a very compliant organ where gene transfer can be stable and successful if provided through an appropriate strategy. The purpose of this review is to present the characteristics of the most efficient virus-derived vectors used by researchers and clinicians to genetically-modify particular cell types or whole regions of the brain. In addition, we discuss major issues regarding side effects such as genotoxicity and immune response associated to the use of these vectorsSuite aux récents succès de divers protocoles thérapeutiques de transfert de gène, notamment appliqués aux pathologies de la rétine, et à la mise sur le marché du Glybera, le premier produit commercial en Europe pour cette classe de médicaments, on observe un regain d'intérêts pour la thérapie génique sur les plans clinique et industriel. Ce nouveau domaine de la médecine expérimentale est particulièrement enthousiasmant si l'on considère que la plupart des maladies neurologiques, attendent désespérément l’apparition d’un traitement satisfaisant. Le système nerveux central est en effet un organe où le transfert de gène peut être stable et réussi s’il est administré selon une stratégie appropriée. L’objectif de cette revue est de présenter les qualités des vecteurs viraux les plus efficaces utilisés actuellement par les chercheurs et les cliniciens pour modifier génétiquement des cellules neurales ou des régions entières du cerveau. Nous abordons également des questions concernant les effets secondaires tels que la génotoxicité et la réponse immunitaire associées à l'utilisation de ces vecteur

Etudes sur la dépréciation, l'amélioration et le contrôle de l'intégration des vecteurs lentiviraux

Integration of HIV vectors in cellular genes creates a risk of insertional mutagenesis and may compromise the safety of the treatment. To counter the risk of insertional mutagenesis studies on abolition or control of the HIV integration have been undertaken. Overall, the efficiencies of these strategies are limited and confined to specific experimental contexts. Therefore, I addressed two issues related to the improvement of HIV vectors: i ) characterization of vectors carrying different mutations of IN, and ii ) the study of self-guided lentiviral particles for targeted integration . Regarding the first part, we compared five mutations representing different functions of the protein. The mutations D64V, Q168A, D167H, N and LQ were chosen. We evaluated the effect of these mutations on the rate of transduction and integration of vectors, and characterized their integration patterns. We observed that the D167H mutation, improves transduction and integration. Other mutants showed a NILV phenotype; mutant D64V allowing greater transduction efficiency and the lowest level of integration and mutation N promoting residual integration in gene -poor regions. In a second project, we have fused cLEDGF with 4 IBDs. These chimeras were fused to the Vpr protein of HIV in order to be packaged into the recombinant particles. We have shown that four chimeras are encapsidated efficiently, they interact with the IN in the capsids, they bind to chromatin when expressed in 293T cells and they provide efficient transduction. Although we do not yet have final results, there are indications that this strategy seems to alter patterns of integration vectors.L intégration des vecteurs dérivés du VIH dans les gènes crée un risque de mutagenèse. Pour y parer, des études d abolition ou de contrôle de l'intégration des vecteurs VIH ont été entreprises. Dans l ensemble, les efficacités de ces stratégies restent limitées et cantonnées à des contextes expérimentaux particuliers. J ai abordé deux sujets relatifs à l amélioration des vecteurs: i) la caractérisation de vecteurs portant différentes mutations de l integrase (IN), et ii) l étude de particules lentivirales autoguidées pour l'intégration ciblée. Concernant la première partie, nous avons comparé 5 mutations de l IN représentatives de différentes fonctions de la protéine. Nous avons évalué l effet de ces mutations sur le taux de transduction et d'intégration des vecteurs, et caractérisé leurs patrons respectifs d'intégration. Nous avons observé que la mutation D167H, améliore la transduction et l'intégration. Les autres mutants ont montré un phénotype de vecteur lentiviral non-intégratif (VLNI); le mutant D64V permettant la plus grande efficacité de transduction et le taux d'intégration le plus faible et la mutation N favorisant l'intégration résiduelle dans des régions pauvres en gène. Dans un deuxième projet, nous avons fusionné le domaine de liaison à l integrase de LEDGF (IBD) à 4 différents domaine de liaison à l ADN (DBD). Ces chimères ont été fusionnées à la protéine Vpr du VIH pour leur encapsidation. Nous avons montré que les 4 chimères sont encapsidées, qu'elles interagissent avec l IN dans les capsides, qu'elles se lient à la chromatine lorsqu'elles sont exprimées dans les cellules 293T et qu elles permettent une transduction efficace. Bien que nous n'ayons pas encore les résultats définitifs, des éléments indiquent que cette stratégie semble modifier les patrons d intégration des vecteurs.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Stratégies de transfert de gène dans le système nerveux central

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Regulatory T Cells Increase After rh-MOG Stimulation in Non-Relapsing but Decrease in Relapsing MOG Antibody-Associated Disease at Onset in Children

International audienceMOGAD. Increase of Th17 cells was significant in MOGNR (means: 0.63 ± 0.15 vs. 1.36 ± 0.43; Wilcoxon-test p = 0.03) but not in MOGR. CD4 + T regs were significantly increased in MOGNR (means: 3.51 ± 0.7 vs. 4.59 ± 1.33; Wilcoxon-test p = 0.046) while they decreased in MOGR. CD45RA-Foxp3 + T regs were significantly decreased in MOGR (means: 2.37 ± 0.23 vs. 1.99 ± 0.17; paired t-test p = 0.021), but not in MOGNR. MOGR showed the highest ratio of effector T regs /non suppressive-T regs, which was significantly higher than in MOGNR. Conclusions: Our findings suggest that CD4 + Th2 and Th17 cells are involved in the pathophysiology of MOGAD in children. The opposite response of T regs to rh-MOG in MOGNR, where CD4 + T regs increased, and in MOGR, where CD45RA-Foxp3 + T regs decreased, suggests a probable loss of tolerance toward MOG autoantigen in MOGR which may explain relapses in this recurrent pediatric autoimmune disease

Comparison Between Several Integrase-defective Lentiviral Vectors Reveals Increased Integration of an HIV Vector Bearing a D167H Mutant

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

Intradermal vaccination prevents anti-MOG autoimmune encephalomyelitis in macaques

International audienceBACKGROUND: Autoimmune demyelinating diseases (ADD) are a major cause of neurological disability due to autoreactive cellular and humoral immune responses against brain antigens. A cure for chronic ADD could be obtained by appropriate immunomodulation.METHODS: We implemented a preclinical scheme to foster immune tolerance to myelin oligodendrocyte glycoprotein (MOG), in a cynomolgus-macaque model of experimental autoimmune encephalomyelitis (EAE), in which administration of recombinant human MOG (rhMOG) elicits brain inflammation mediated by MOG-autoreactive CD4+ lymphocytes and anti-MOG IgG. For immunotherapy, we used a recombinant antibody (Ab) directed against the dendritic cell-asialoglycoprotein receptor (DC-ASGPR) fused either to MOG or a control antigen PSA (prostate-specific antigen).FINDINGS: rhMOG and the anti-DC-ASGPR-MOG were respectively detected in CD1a+ DCs or CD163+ cells in the skin of macaques. Intradermal administration of anti-DC-ASGPR-MOG, but not control anti-DC-ASGPR-PSA, was protective against EAE. The treatment prevented the CD4+ T cell activation and proinflammatory cytokine production observed in controls. Moreover, the administration of anti-DC-ASGPR-MOG induced MOG-specific CD4+CD25+FOXP3+CD39+ regulatory lymphocytes and favoured an upsurge in systemic TGFβ and IL-8 upon rhMOG re-administration in vivo.INTERPRETATION: We show that the delivery of an anti-DC-ASGPR-MOG allows antigen-specific adaptive immune modulation to prevent the breach of immune tolerance to MOG. Our findings pave the way for therapeutic vaccines for long-lasting remission to grave encephalomyelitis with identified autoantigens, such as ADD associated with anti-MOG autoantibodies. FUND: Work supported by the French ANR (ANR-11-INBS-0008 and ANR-10-EQPX-02-01), NIH (NIH 1 R01 AI 105066), the Baylor Scott and White Healthcare System funding and Roche Research Collaborative grants