Vaccination thérapeutique de macaques cynomolgus contre le virus VISmac251 (stock 11/88) (un modèle pour l' immunothérapie humaine anti-VIH ?)

PARIS-BIUSJ-Thèses (751052125) / SudocSudocFranceF

Lentiviral-mediated gene transfer to model triplet repeat disorders

This chapter describes the potential use of viral-mediated gene transfer in the central nervous system as a new strategy in developing animal models of neurodegenerative diseases. To illustrate the approach, procedures for the production of lentiviral vectors encoding polyQ proteins are provided, as well as methods for the determination of viral titers, in vitro infection, and basic protocols for in vivo studies in rodents

Early and reversible neuropathology induced by tetracycline-regulated lentiviral overexpression of mutant huntingtin in rat striatum

International audienc

Continuous delivery of human and mouse erythropoietin in mice by genetically engineered polymer encapsulated myoblasts

The transplantation of polymer encapsulated myoblasts genetically engineered to secrete erythropoietin (Epo) may obviate the need for repeated parenteral administration of recombinant Epo as a treatment for chronic renal failure, cancer or AIDS-associated anemia. To explore this possibility, the human and mouse Epo cDNAs under the control of the housekeeping mouse PGK-1 promoter were transfected into mouse C2C12 myoblasts, which can be terminally differentiated upon exposure to low serum-containing media. Pools releasing 150 IU human Epo per 10(6) cells per day and 390 IU mouse Epo per 10(6) cells per day were selected. Polyether-sulfone (PES) capsules loaded with approximately 200,000 transfected myoblasts from these pools were implanted on the dorsal flank of DBA/2J, C3H and C57BL/6 mice. With human Epo secreting capsules, only a transient increase in the hematocrit occurred in DBA/2J mice, whereas no significant response was detected in C3H or C57BL/6 mice. On the contrary, all mice implanted with capsules releasing mouse Epo increased their hematocrit over 85% as early as 7 days after implantation and sustained these levels for at least 80 days. All retrieved implants released Epo and contained well preserved myoblasts. Moreover most capsules were surrounded by a neovascularization. Mice transplanted with nonencapsulated C2C12 cells releasing mouse Epo showed only a transitory elevation of their hematocrit reflecting the poor engraftment of injected myoblasts. These results indicate that polymer encapsulation of genetically engineered myoblasts is a promising approach for the long-term delivery of bioactive molecules, allowing the resolution of the shortcomings of free myoblast transfer

A gene therapy approach to regulated delivery of erythropoietin as a function of oxygen tension

Current therapy for several forms of anemia involves a weekly regime of multiple subcutaneous injections of recombinant human erythropoietin (hEpo). In an effort to provide a physiologically regulated administration of erythropoietin, we are developing cell lines genetically engineered to release hEpo as a function of oxygen tension. C2C12 cells were transfected using a vector containing the hEpo cDNA driven by the hypoxia-responsive promoter to the murine phosphoglycerate kinase gene. In vitro, these cells showed a threefold increase in hEpo secretion as oxygen levels were shifted from 21% to 1.3% oxygen. To test in vivo response, C2C12-hEpo cells were encapsulated in a microporous membrane and implanted subcutaneously on the dorsal flank of DBA/2J mice. On average, serum hEpo levels in animals exposed to 7% oxygen were two-fold higher than values seen in their control counterparts kept at 21% oxygen. Similar studies employing rats confirmed that hEpo delivery is regulated as a function of oxygen tension. These results suggest the feasibility of developing an oxygen-regulated, encapsulated cell-based system for hEpo delivery

Inhibition of calcineurin by FK506 protects against polyglutamine-huntingtin toxicity through an increase of huntingtin phosphorylation at S421

Huntington's disease (HD) is caused by an abnormal expanded polyglutamine (polyQ) repeat in the huntingtin protein. Insulin-like growth factor-1 acting through the prosurvival kinase Akt mediates the phosphorylation of huntingtin at S421 and inhibits the toxicity of polyQ-expanded huntingtin in cell culture, suggesting that compounds enhancing phosphorylation are of therapeutic interest. However, it is not clear whether phosphorylation of S421 is crucial in vivo. Using a rat model of HD based on lentiviral-mediated expression of a polyQ-huntingtin fragment in the striatum, we demonstrate here that phosphorylation of S421 is neuroprotective in vivo. We next demonstrate that calcineurin (CaN), a calcium/calmodulin-regulated Ser/Thr protein phosphatase, dephosphorylates S421 in vitro and in cells. Inhibition of calcineurin activity, either by overexpression of the dominant-interfering form of CaN or by treatment with the specific inhibitor FK506, favors the phosphorylation of S421, restores the alteration in huntingtin S421 phosphorylation in HD neuronal cells, and prevents polyQ-mediated cell death of striatal neurons. Finally, we show that administration of FK506 to mice increases huntingtin S421 phosphorylation in brain. Collectively, these data highlight the importance of CaN in the modulation of S421 phosphorylation and suggest the potential use of CaN inhibition as a therapeutic approach to treat HD

Akt is altered in an animal model of Huntington's disease and in patients

The insulin-like growth factor I (IGF-1)/Akt pathway plays a crucial role in Huntington's disease by phosphorylating the causative protein, polyQ-huntingtin, and abolishing its toxic properties [Humbert et al. (2002)Dev. Cell, 2, 831-837; Rangone et al. (2004)Eur. J. Neurosci., 19, 273-279]. Therefore, dysregulation of this pathway may be essential for disease progression. In the present report, we thus aimed to analyse the status of Akt in brain or in peripheral tissues in Huntington's disease. Using a genetic model of Huntington's disease in rat that reproduces neuronal dysfunction and death, we show a progressive alteration of Akt during neuronal dysfunction and prior neurodegeneration. By analysing a limited number of lymphoblasts and lymphocytes, we detected modifications of Akt in Huntington's disease patients confirming a dysregulation of Akt in the disease process. Finally, we demonstrate that during late stages of the disease, Akt is cleaved into an inactive form by caspase-3. These observations demonstrate a progressive but marked alteration of this pro-survival pathway in Huntington's disease, and further implicate it as a key transduction pathway regulating the toxicity of huntingtin

CA150 rescues striatal cell death in overexpression and knock-in models of Huntington's disease

International audienceHuntington’s disease (HD) is a dominant neurodegenerative disorder associated with N-terminal expanded polyglutamines (polyGlns) in huntingtin (htt). The abnormal regulation of transcription may be central to HD neuronal pathology. We previously reported that the transcriptional regulator CA150 binds to htt and accumulates in a neuropathological grade dependent manner in the striatal and cortical neurons of HD patients, suggesting CA150 dysfunction in HD. Consistently, we report herein that CA150 overexpression rescued the toxicity of mutant htt in lentiviral overexpression (rat striatum) and knock-in (striatal cells from HdhQ111 mice) models of HD. In both systems, the rescue was dependent on the presence of the (Gln-Ala)38 repeat normally found in CA150. Our data indicate that the striatal pathology in HD may involve a loss of CA150 function. Additionally, our data provide a biological rationale for the previously reported influence of the enlarged and potentially hypomorphic repeat alleles on the age at onset in H