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  Director : HEARD Jean Michel (: jmheard@pasteur.fr)


  abstract

 

One topic of the laboratory is the development of strategies for gene therapy. Investigated models concerns the nervous system and the erythropoiesis. Vectors derived from lentiviruses or AAV are used. The laboratory is also interested in cell-retrovirus interactions. Studies are focused on very early stages of virus entry, antigen presentation and the role of the HIV Nef protein.



  report

cale

Gene therapy to the nervous system.

Lysosomal storage diseases can be corrected through the delivery of the missing enzyme. For those diseases affecting the central nervous system, which are the more frequent ones, intracerebral delivery supposes in situ enzyme secretion. This can be obtained by gene therapy methods. Strereotactic injection of AAV-based or lentivirus-based vectors encoding the missing enzyme in the brain leads to inducing enzyme secretion in a small number of genetically-modified cells that provide an intra-cerebral source of enzyme. Enzyme can be transported to remote locations and complete correction of storage lesions is observed in the entire brain. We obtained these results in the mouse model of MPS VII, which is deficient for ß-glucuronidase, and in the MPS I mouse, which is deficient for a-iduronidase and which provides a model for Hurler's disease, a disorder relatively frequent in children. With the support of the AFM, we have installed a colony of dogs with Hurler's disease in France and we currently initiate a study of the efficiency of the approach in affected puppies. The immediate goal is to assess the feasibility of the method for the treatment of children and to provide information for a discussion of the opportunity of a therapeutic trial (collaboration with M. Tadié, Laboratoire de Neurochirurgie, CHU Bicêtre and P. Moullier, CHU Hôtel-Dieu, Nantes).

Spinal cords injuries are frequent and responsible of devastating conditions in young trauma patients. We have initiated this year a program aimed at exploring a reconstruction method based on the association of microsurgery and gene therapy after spinal cord section in rats. Surgery allows to reconnect motor neurons located above the section with hind limb muscles, and eliminates all connections with the distal spinal cord stump. A regain of motricity is observed, however, movements are not adapted to locomotion. This experimental model is used to assess the efficiency of the transfer of genes coding for factors involved in neuron survival, spinal cord plasticity and axonal growth (S. Liu, D. Bohl).

Gene therapy for stimulating erythropoiesis.

The goal of this program is to stimulate fetal erythropoiesis, in which hemoglobin structure is a 2/g 2, in ß-thalassemic patients who are deficient for the synthesis of the ß-chain. Strategy is based on the prolonged induction of a stress erythropoiesis, which is mostly of the fetal type, through the delivery of large amounts of erythropopietin (Epo). Intense erythropoietic stimulation hastens the differentiation of early erythroid progenitors, which escape silencing of the g gene. We have described this mechanism and validated the strategy in ß-thalassemic mice by transferring the mouse Epo cDNA into skeletal muscles. Disease correction was demonstrated after naked DNA electrotransfer and with AAV vectors in which Epo expression level could be controlled by tetracycline. This non-expensive and simple method, which appears safe, at least when naked DNA is used, could be appropriate for the treatment of millions of ß-thalassemic patients who, for most of them, leave in developing countries, especially in South-East Asia (S. Samakoglou, D. Bohl).

 

Topics on early stages of retroviral infection

Topology and oligomerization of Pit-2, a retroviral receptor and a phosphate transporter

PiT-2 functions as the cell surface anchor molecule for retroviral particles bearing an amphotropic envelope and mediates fusion following conformational changes of the envelope-receptor complex. PiT-2 also transports phosphate and sodium. Both activities vary inversely with extracellular inorganic phosphate concentrations. Variation is rapid and occurs in the absence of protein synthesis. PiT-2 is a multi-transmembrane and highly hydrophobic protein. We have shown that PiT-2 belongs to a large family represented in the various reigns. All members contain a duplication of the conserved domain. We have defined the topologic organization of PiT-2 and shown that the duplicated domains are in opposite topology with respect to the plasma membrane. Using cross-linking reagents, we showed that PiT-2 formed assemblies at the cell surface, and that these assemblies underwent conformational changes in response to variation of extracellular phosphate concentration. The compacted form is associated with high biological activity, whereas the relaxed form has little or no activity (C. Salaün, P. Rodrigues).

Works by the team of Olivier Schwartz

HIV principally infects T lymphocytes and antigen-presenting cells like macrophages and dendritic cells. Infection requires a fusion of the viral and plasma membranes for releasing capsids into the cytosol, where the subsequent stages of the viral cycle must take place. Fusion involves interaction between the envelope glycoprotein and cell surface receptors. Macrophages, and in a slightly different way, dendritic cells, are able to capture viral particles in intracellular vesicles notwithstanding envelope/receptor interactions. We have shown that in macrophages, these vesicles are related to macropinocytic vesicles and suggested that fusion may also take place in these structures (V. Maréchal).

In dendritic cells, the capture of viral particles is favored by a lectin called DC-SIGN. Intra-vesicular particles keep infectivity and can be transmitted to CD4 target cells during antigen presentation. We have shown that the expression of DC-SIGN at the cell surface is upregulated when the HIV-1 Nef protein is expressed. DC-SIGN up-regulation in infected cells allows the capture of increased numbers of HIV particles and leads to a more efficient transmission of the infection to target cells. Mechanisms involved in the action of Nef on DC-SIGN have been elucidated. They consist in a modification of the intracellular trafficking of DC-SIGN, which relies on an endocytosis motif similar to that previously recognized as a target for Nef action on CD4 (N. Sol-Foulon, C. Nobile).

The release of HIV particles in the cytosol after membrane fusion is followed by the disassembly and degradation of capsid proteins. We have now provided evidence that peptides generated during this process can join MHC-I molecules and be presented to specific CTLs at the cell surface (O. Schwartz, collaboration with F. Buseyne). This antigen presentation through the exogenous pathway potentially plays a role in the antiviral immune response. We are currently examining the molecular and cellular mechanisms involved in this phenomenon. We are studying what are the routes of entry and trafficking of virions leading to exogenous presentation of epitopes by MHC-I. Our aim is also to compare the repertoire of viral epitopes presented through the exogenous pathway or after neo-synthesis of viral proteins by the infected cell (A. Morris).



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

Renée Communal, communal@pasteur.fr

Olivier Schwartz, CR Institut Pasteur, schwartz@pasteur.fr

Delphine Bohl, CR2 INSERM, dbohl@pasteur.frs

Nathalie Sol-Foulon, CR2 CNRS, natsol@pasteur.fr

Arnaud Moris, Stage post-doctoral, moris@paseur.fr

Song Liu, Stage post-doctoral, songliu@pasteur.fr

Christine Salaün, Thèse Ecole Doctorale B2M, csalaun@pasteur.fr

Emmanuel Gyan, Etudiant DEA, egyan@pasteur.fr

Cinzia Nobile, Etudiante étrangère, nobileci@pasteur.fr

Valérie Maréchal, Technicienne Supérieure 2D, marechal@pasteur.fr

Nathalie Desmaris, Technicienne Supérieure 1D, nchodan@pasteur.fr


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