INDUCTION DE TOLERANCE SPECIFIQUE DU TRANSGENE APRES THERAPIE GENIQUE – GENETOL

Several vector systems allow efficient gene transfer and transgene expression in vivo. Recombinant adeno-associated vector (rAAV) is one of them and an increasing number of reports demonstrate that rAAV administered in situ in many different target organs can sustain permanent gene expression after one single administration. However, it appears that when translational studies are initiated in larger animal species, the expression of heterologous and even autologuous proteins is systematically followed by a deleterious and often destructive immune reaction from the host. This was shown in cat, dog and nonhuman primate models. More recently, the involvement of the immune system towards the rAAV capsid itself was also reported in a small cohort of factor IX-deficient patients. Therefore, if rAAV provides a clinically relevant platform for efficient and sustained gene therapy, understanding what factors trigger the host immune system remains a major issue before moving to humans. Also, the development of appropriate and relevant immunosuppression protocols is warranted to optimize clinical translation. Long-term use of immunosuppressive regiments is often associated with severe adverse effects, which may be an important limitation. Aside from investigating the clinical impact of new immunosuppressor generations, it appears necessary to understand the mechanisms responsible for triggering the immune system in the context of gene transfer in the skeletal muscle as well as to develop strategies inducing tolerance to the transgene and/or visual capsid. One of the difficulties is to address this question in a relevant animal model that mimics to some extent the patient's immune system. We believe that in that respect, the non-human primate (NHP) is a unique model that is complementary to what the murine model provides. Combining the expertise of Inserm UMR 649 in rAAV-based gene transfer in NHP with the one of Inserm UMR643 in tolerance induction strategies, we identified the following aims: 1) Given that the human and primate immune systems are different from that of the mouse, we propose to characterize the priming events responsible for the initiation of the host immune response against the transgene and/or the capsid following intramuscular (IM) rAAV delivery in the clinically relevant macaque model. 2) As an alternative to the immunogenic IM route, we have developed in NHP a strategy based on the regional perfusion (RI) of an isolated limb. Our preliminary data demonstrate that the RI route is less immunogenic against the transgene than the IM route. We propose therefore to dissect the mechanisms of transgene tolerance following RI rAAV delivery. 3) Also, we aim to develop a protocol to induce targeted and specific immune transgene tolerance following rAAV IM delivery in order to achieve sustained transgene expression without compromising the patient's immune status. Recent studies, performed by Inserm UMR 643 and Inserm UMR 649 together, have demonstrated that immature dendritic cells (DC) could be potential reagents to promote antigen-specific tolerance in vivo. We propose to test the tolerogenic effect of these cells in the macaque model of following rAAV-delivery. Altogether, the data emerging from the overall project will contribute to the development of optimal and safe rAAV-based gene therapy strategies for patients with genetic muscular disorders, as well for the treatment of several genetic indications requiring the secretion of therapeutic levels of circulating factors.