Contributions du Laboratoire Conchylicole de Poitou-Charente (LCPC) au défi MOREST (mortalités estivales d'huîtres creuses) en 2003. Thématique "Ecologie côtière"

Depuis 2001, le laboratoire participe au défi MOREST, comme laboratoire d'Ecologie côtière dans l'organisation et l'animation d'études pluridisciplinaires et comme site atelier du défi. En 2003, le LCPC encadre une opération de recherche impliquant 14 laboratoires sur l'étude du modèle de mortalité plat table dans le Bassin de Marennes Oléron. Un large panel des causes de mortalité est étudié; facteurs physico chimiques, agents pathogènes, substances toxiques (pesticides ou relargages chimiques), etc …. Le labo est également impliqué en accompagnement d'actions de caractérisation de la mortalité de naissain (lignées consanguines sur estran) et sur des opérations de caractérisation précoce de la mortalité en laboratoire. Enfin, l'effet "site de gestion hivernale" sur la survie des huîtres en année (n+1) est testé sur le site atelier du Bassin de Marennes Oléro

Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

International audienceClinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field

Lack of an Immune Response against the Tetracycline-Dependent Transactivator Correlates with Long-Term Doxycycline-Regulated Transgene Expression in Nonhuman Primates after Intramuscular Injection of Recombinant Adeno-Associated Virus

We previously documented persistent regulation of erythropoietin (Epo) secretion in mice after a single intramuscular (i.m.) injection of a recombinant adeno-associated virus (rAAV) vector harboring both the tetracycline-dependent transactivator (rtTA) and the Epo cDNA (D. Bohl, A. Salvetti, P. Moullier, and J. M. Heard, Blood 92:1512-1517, 1998). Using the same vector harboring the cynomolgus macaque Epo cDNA instead, the present study evaluated the ability of the tetracycline-regulatable (tetR) system to establish long-term transgene regulation in nonhuman primates. The vector was administered i.m., after which 5-day induction pulses were performed monthly for up to 13 months by using doxycycline (DOX), a tetracycline analog. We show that initial inductions were successful in all individuals and that there was a tight regulation and a rapid deinduction pattern upon DOX withdrawal. For one macaque, regulation of Epo secretion was maintained during the entire experimental period; for the five remaining macaques, secreted Epo became indistinguishable from endogenous Epo upon repeated DOX inductions. We investigated the mechanism involved and showed that, except in the animal in which secretion persisted, delayed humoral and cellular immune responses were directed against the rtTA transactivator protein associated with the reduction of vector DNA in transduced muscles. This study provides some evidence that, when the immune system is not mobilized against the rtTA transactivator, the tetR-regulatable system is able to support long-term transgene regulation in the context of an rAAV in nonhuman primates. In addition, our results suggest potential improvements for vector design