Preclinical gene therapy studies, altered lymphocyte development and function in ADA-SCID

Genetic defects in the adenosine deaminase (ADA) gene are among the most common causes for severe combined immunodeficiency (SCID). ADA-SCID patients suffer from lymphopenia, absent cellular and humoral immunity, recurrent infections and autoimmune manifestations in milder forms. Currently available therapeutic options for this otherwise fatal disorder include bone marrow transplantation (BMT), enzyme replacement therapy with bovine ADA (PEG-ADA) or hematopoietic stem cell gene therapy (GT). The overall aims of my this thesis were to evaluate the preclinical safety of HSC gene therapy and the basis involved in autoimmune manifestations observed in ADA deficiency. For the first part of the project I assessed the feasibility to perform two preclinical studies recommended by Regulatory Authorities: toxicology/tumorigenicity and biodistribution studies. Both studies were performed using the NSG immunodeficient mouse model transplanted with murine or human hematopoietic stem/progenitor cells transduced with a Retroviral vector with an amphotrophic envelope encoding for ADA. For the toxicology/tumorigenicity study we tested different transduction protocols both in vitro and in vivo showing a low transduction efficiency in ADA-/- mouse lineage negative cells. These results led us to conclude that this model is not suitable for in vivo toxicology/tumorigenicity studies. On the other hand, transduction efficiency in human hematopoietic stem/progenitor cells was higher compared to murine cells. Biodistribution study with human CD34+ cells derived from cord blood showed a good engraftment of human cells in NSG hosts with transduced cells in PB and lymphoid organs, in line with the frequency found in treated patients.The second part of my PhD project aimed at investigating Treg function and B cell development in ADA-deficient mice. Autoimmune manifestations including type I diabetes, hypothyroidism, autoimmune thrombocytopenia, and haemolytic anaemia are frequently observed in the ADA-SCID patients treated with PEGADA, BMT and GT. We investigated the mechanisms which might be involved in these alterations. We found that PEG_ADA treated mice represent a model to study autoimmunity as they developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Moreover, Tregs isolated from PEG-ADA– treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. mice showed a mild alteration in the bone marrow and splenic B cell subsets. We also explored whether an increased egress of immature and recirculant B cells from the bone marrow through the synovial vessels due to the activation of endocannabinoid pathway might contribute to autoimmune manifestations in this disease. B-cell escaping central tolerance mechanisms together with nonfunctional Treg cells in the periphery might further accelerate the onset of autoimmunity. In summary results of this work have contributed to improved our knowledge on ADA-SCID and facilitate the progress of clinical development of gene therapy for ADA-SCID.