In vivo induction of Ag-specific tolerance by hepatocyte-targeted gene transfer

Lentiviral vectors (LV) are widely used vehicles for gene therapy in pre-clinical animal models and clinical trials with promising safety and efficacy results. However, immune responses directed towards LV, transgene (Tg) product, or both may limit the efficacy of gene therapy. To overcome this limitation the LV.ET.142T platform has been developed and effectively applied for gene replacement therapy in hemophilia B or for promoting/restoring tolerance in autoimmune diabetes, sustaining hepatocyte-targeted gene transfer as a novel way to promote tolerance. In the present research we are planning to broaden application of the LV.ET.142T platform and improving its efficacy. Specifically, we will investigate the ability of LV.ET.142T to induce tolerance to alloAgs. The efficacy combined therapy of the LV.ET.142T encoding for the insulin B chain 9-23 (LV.ET.InsB9-23.142T) and anti-CD3 mAb in controlling recurrence of autoimmunity in NOD mice following autologous and allogeneic pancreatic islet transplant will be investigated. Several factors may negatively impact the efficacy of LV.ET.142T: the leakiness of transgene expression, the type of transgene and the pre-existing inflammatory immune environment arising from a genetic predisposition. IDUA knockout mouse, the model of mucopolysaccaridosis I, is particularly resistant to tolerance induced by LV.ET.142T, thus it represents a valuable model to better understand how transgene immunity occurs after gene therapy and test optimized vectors with additional layers of transgene regulation and/or combined therapies finalized to pre-tolerize the recipient prior gene therapy. The results obtained in this project will strengthen the LV.ET.142T platform as an inverse vaccination strategy and broaden its application beyond gene replacement in monogenic diseases and towards the treatment T-cell mediated diseases.