DAISY

In solid organ transplantation, immune incompatibility between donor and recipient leads to a vigorous immune response against the graft, entailing rejection in absence of treatment. Prevention of graft rejection requires long-term administration of immunosuppressive drugs that dampen all immune responses, resulting in high rates of infections and malignancies. We propose to develop another strategy, targeting specifically the alloimmune response, based on the engineering of recipient’s regulatory T cells through gene transfer and editing. More specifically, forced expression of a donor HLA-targeted Chimeric Antigen Receptor (CAR) can redirect human regulatory T cells against an allograft. Briefly, human regulatory T cells are sorted out, activated and then transduced with a viral vector encoding donor HLA-targeted CAR, resulting in the generation of CAR-Tregs. Following an in vitro expansion phase, the cell product is tested to ensure Treg lineage stability, and then infused to humanized mice using preclinical models of transplantation. Our preliminary data demonstrate in vitro and in vivo efficacy of CAR-Tregs in controlling the alloimmune response. We showed their ability at preventing acute graft-versus-host disease (GVHD), induced by the transfer of human mature T lymphocytes into immunocompromised mice. Next, we aim to evaluate their potential in the prevention of chronic rejection and in the achievement of immune tolerance, using more sophisticated models that allow to study long-term functional and histological graft outcomes. CAR-Tregs therapy will be worth its cost only if it provides a breakthrough and allows to get rid of long-term immunosuppression-related side effects. To this end, the research project focus on two main axis: 1- The first axis aims to optimize a combined therapeutic strategy that exploits the synergy effects of highly-targeted immunotherapies and donor-specific CAR-Treg. We propose to make CAR-Tregs resistant to an immunotherapy through Crispr/Cas9 genome editing. Administration of this immunotherapy will therefore target exclusively effector cells involved in the rejection response, while sparing CAR-Tregs, and therefore further tip the balance toward regulation in vivo. Further, the strategy will reduce the number of CAR-Tregs required to elicit a therapeutic effect. The shorter the manufacturing process, the greater Treg stability and longevity. 2- The second axis aims to generate highly-relevant preclinical models, based on full humanized immune system mice, through the transfer of human hematopoietic stem and progenitor cells in neonates. These humanized mice do not develop GVHD, since human cells are educated in mouse lymphoid organs, and allow to investigate the ability of CAR-Tregs to protect from chronic rejection in skin and islet transplantation models. This validation step in highly relevant preclinical models is mandatory on the path toward clinical implementation.