468 glp compliant non clinical safety and biodistribution of a recombinant aav2 8 vector administered intravenously for treatment of mucopolysaccharidosis type vi

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disorder caused by deficiency of the enzyme arylsulfatase B (ARSB), which results in widespread accumulation and excretion of toxic glycosaminoglycans. We recently developed a successful gene therapy approach based on a single systemic administration of AAV2/8 that targets liver of MPS VI animal models. In view of a gene therapy clinical trial for MPS VI, we performed GLP-compliant non-clinical studies to assess the safety and biodistribution of AAV2/8. TBG. hARSB, a recombinant AAV2/8 vector encoding human ARSB (hARSB) under the control of the thyroxine-binding globulin promoter (TBG). We used transgenic C57/BL6-TgARSBC91S mice that overexpress an inactive hARSB C91S mutant and are thus immune tolerant to hARSB. Mice were treated with either AAV2/8.TBG. hARSB or the vehicle alone, as control. Toxicity was evaluated on day 15 (D15) and 180 (D180) after systemic injection of 2×1013 gc/kg, which is 10X the highest dose proposed for the clinical study [20males(M)+20females(F)/treatment/timepoint]. No mortality, abnormal clinical signs and alteration in body weight, body temperature and food intake were observed through the study. Similarly, no clinically relevant changes in blood chemistry and hematology were found in treated mice compared to controls. Histopathology revealed thyroid epithelial hypertrophy in AAV-treated mice. AAV2/8.TBG. hARSB biodistribution and expression was evaluated on D15 and D180 at the dose of 2×1012 gc/kg, which is 1X the highest dose proposed for the clinical study (5M+5F/treatment/timepoint). Although vector DNA was present in all organs on D15, it was sequestered mainly in liver at levels at least 3 logs higher than those found in other organs. Vector DNA declined on D180, but remained high in liver. Accordingly, hARSB was mainly expressed stably in liver, supporting TBG tissue specificity. Vector DNA was found in gonads of both sexes at 3 logs lower than in liver. A robust reduction of vector DNA was observed on D180. A supportive study conducted in male rabbits showed that vector shedding in semen was only transient, which suggests that the risk of inadvertent germline transmission of AAV2/8. TBG.hARSB is minimal at least in male animals. An in situ hybridization study is ongoing in ovaries to elucidate AAV localization. Finally, AAV DNA was only transiently present in plasma, urine and stools of mice (up to D37, D2 and D14, respectively), which minimizes the potential risk associated with transmission to third parties and/or the environment. In conclusion, these studies show a safe profile of intravenous administrations of AAV2/8. TBG.hARSB and pave the way for the phase I/II clinical trial

Concomitant heterochromatinisation and down-regulation of gene expression unveils epigenetic silencing of RELB in an aggressive subset of chronic lymphocytic leukemia in males

<p>Abstract</p> <p>Background</p> <p>The sensitivity of chronic lymphocytic leukemia (CLL) cells to current treatments, both <it>in vitro </it>and <it>in vivo</it>, relies on their ability to activate apoptotic death. CLL cells resistant to DNA damage-induced apoptosis display deregulation of a specific set of genes.</p> <p>Methods</p> <p>Microarray hybridization (Human GeneChip, Affymetrix), immunofluorescent <it>in situ </it>labeling coupled with video-microscopy recording/analyses, chromatin-immunoprecipitation (ChIP), polymerase chain reactions (PCR), real-time quantitative PCR (RT-QPCR) and bisulfite genome sequencing were the main methods applied. Statistical analyses were performed by applying GCRMA and SAM analysis (microarray data) and Student's t-test or Mann & Whitney's U-test.</p> <p>Results</p> <p>Herein we show that, remarkably, in a resistant male CLL cells the vast majority of genes were down-regulated compared with sensitive cells, whereas this was not the case in cells derived from females. This gene down-regulation was found to be associated with an overall gain of heterochromatin as evidenced by immunofluorescent labeling of heterochromatin protein 1α (HP-1), trimethylated histone 3 lysine 9 (3metH3K9), and 5-methylcytidine (5metC). Notably, 17 genes were found to be commonly deregulated in resistant male and female cell samples. Among these, <it>RELB </it>was identified as a discriminatory candidate gene repressed in the male and upregulated in the female resistant cells.</p> <p>Conclusion</p> <p>The molecular defects in the silencing of <it>RELB </it>involve an increase in H3K9- but not CpG-island methylation in the promoter regions. Increase in acetyl-H3 in resistant female but not male CLL samples as well as a decrease of total cellular level of RelB after an inhibition of histone deacetylase (HDAC) by trichostatin A (TSA), further emphasize the role of epigenetic modifications which could discriminate two CLL subsets. Together, these results highlighted the epigenetic <it>RELB </it>silencing as a new marker of the progressive disease in males.</p

Etude des interactions gène-environnement dans la régulation de la pression artérielle (E-sélectine et P-sélectine)

L'hypertension est un facteur de risque cardiovasculaire important. Après avoir rassemblé les gènes et leurs polymorphismes susceptibles de moduler l'hypertension, y compris ceux impliqués en pharmacogénétique, nous avons approfondi dans ce travail un groupe de gènes et de leurs produits, les sélectines. Les sélectines (E-sélectine, P-sélectine et L-sélectine) sont des molécules d'adhésion localisées à la surface des cellules endothéliales activées et en surface des leucocytes. Elles participent à l'étape initiale du recrutement des leucocytes circulants qui précède leur migration vers la zone inflammatoire. Les sélectines sont impliquées dans de nombreuses pathologies parmi lesquelles les maladies cardio-vasculaires, les maladies inflammatoires et auto-immunes. Certains polymorphismes génétiques de la E-sélectine et de la P-sélectine sont associés à ces diverses pathologies. De même, les concentrations sanguines de ces molécules, reflet de leur expression cellulaire, sont augmentées en conditions pathologiques. Au sein d'une large population supposée saine (Cohorte Stanislas) nous avons montré que les facteurs environnementaux tels que l'âge, le sexe, la surcharge pondérale, et l'alcool sont susceptibles de modifier l'impact de polymorphismes de la E-sélectine sur la définition des niveaux de pression artérielle. Dans un second temps, nous avons étudié l'influence des polymorphismes génétiques de la E-sélectine et de la P-sélectine sur leur taux circulants. Bien qu'aucun des polymorphismes G98T, Serl28Arg et Leu554Phe, n'influencent le taux de E-sélectine, nous avons montré que les polymorphismes 599Val/Leu, 715 Thr/Pro, et N562D du gène de la P-sélectine influencent fortement la concentration en P-sélectine. Dans un troisième temps nous avons démontré qu'il existe des ressemblances familiales des concentrations en E-sélectine et P-sélectine, suggérant un rôle majeur de la génétique et de l'environnement partagé dans la régulation des concentrations de ces molécules d'adhésion. Enfin, nous avons débuté des études de transcriptomique et pour ce faire nous avons réalisé une banque de sang et de lymphocytes. L'ensemble de ces résultats permet de mieux comprendre la régulation physiologique de la E- sélectine et de la P-sélectine ainsi que les facteurs à prendre en compte lors d'études cliniques et pharmacologiques impliquant ces molécules. L'approche transcriptomique devrait par ailleurs renforcer la connaissance actuelle du lien entre le gène et la protéine.NANCY1-SCD Pharmacie-Odontologie (543952101) / SudocSudocFranceF

Pharmacogenomics and antihypertensive drugs: a path toward personalized medicine

International audienc

“RCL-Pooling Assay”: A Simplified Method for the Detection of Replication-Competent Lentiviruses in Vector Batches Using Sequential Pooling

International audienc

464. RCL-Pooling Assay: A Simplified Method for the Detection of Replication Competent Lentiviruses in Vector Batches Using Sequential Pooling

International audienc