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

mRNA COVID-19 vaccine booster fosters B- and T-cell responses in immunocompromised patients

SARS-CoV-2 vaccination has proven effective in inducing an immune response in healthy individuals and is progressively us allowing to overcome the pandemic. Recent evidence has shown that response to vaccination in some vulnerable patients may be diminished, and it has been proposed a booster dose. We tested the kinetic of development of serum antibodies to the SARS-CoV-2 Spike protein, their neutralizing capacity, the CD4 and CD8 IFN-γ T-cell response in 328 subjects, including 131 immunocompromised individuals (cancer, rheumatologic, and hemodialysis patients), 160 health-care workers (HCW) and 37 subjects older than 75 yr, after vaccination with two or three doses of mRNA vaccines. We stratified the patients according to the type of treatment. We found that immunocompromised patients, depending on the type of treatment, poorly respond to SARS-CoV-2 mRNA vaccines. However, an additional booster dose of vaccine induced a good immune response in almost all of the patients except those receiving anti-CD20 antibody. Similarly to HCW, previously infected and vaccinated immunocompromised individuals demonstrate a stronger SARS-CoV-2–specific immune response than those who are vaccinated without prior infection

Alterations in the brain adenosine metabolism cause behavioral and neurological impairment in ADA-deficient mice and patients

Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency