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Pathogenesis and therapy of primary immunodeficiencies

SR-Tiget Unit
Alessandro Aiuti, Clinical Research Coordinator and Head of Unit

Primary immunodeficiencies are a group of rare genetic diseases characterized by an altered innate and adaptive immune system, increased susceptibility to infections, risk of autoimmunity and cancer. Adenosine deaminase (ADA)- deficient severe combined immunodeficiency (SCID) is a complex metabolic and immunological disorder, characterized by a severe immunodeficiency due to the accumulation of purine metabolites in plasma and cells. Without treatment, the condition is fatal and requires early intervention. Our studies are aimed at investigating the molecular and cellular mechanisms linking the purine metabolism disorder to the immune and non-immune alterations of the disease. SR-Tiget was one of the pioneers in bringing gene therapy with retrovirally transduced hematopoietic stem cells from preclinical studies to successful clinical applications. Studies on vector integrations are providing crucial information on vector biology, the dynamics of genetically modified hematopoietic stem cells, and the safety of gene therapy. In addition, we are developing new therapeutic strategies based on the use of lentiviral vectors for ADA-SCID and other primary immunodeficiencies, including Wiskott-Aldrich Syndrome and Chronic Granulomatous Disease. These studies will contribute to improve our knowledge on primary immunodeficiencies and improve patients’ care.

Research activities (updated 2015)


Adenosine deaminase (ADA)-SCID is characterized by impaired lymphocyte development and function, as well as systemic organ damage due to metabolic toxicity. Haematopoietic stem cell transplantation is the major treatment for ADA-SCID, although survival following different donor sources varies considerably. Unlike other SCID forms, 2 other options are available for ADA-SCID: enzyme replacement therapy PEG-ADA, and autologous haematopoietic stem cell gene therapy. ADA-deficient patients with late-onset forms or after treatment are known to manifest immune dysregulation. Our aim is to acquire new information on the autoimmune manifestations of this disease and to investigate the ability of different treatments to correct them. Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. We obtained evidence that adenosine, accumulating in the absence of ADA and its excessive turn-over by PEG-ADA interfere with Treg function. Tregs isolated from PEG-ADA treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. PEG-ADA treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The observed loss of function in ADA-deficient Tregs provides new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.

To assess whether ADA deficiency affects the establishment of B cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells of ADA-SCID patients before and after HSC-GT. We found that before HSC-GT, B cells from ADA-SCID patients contained more autoreactive and ANA-expressing clones, indicative of defective central and peripheral B-cell tolerance checkpoints. Strikingly, after HSC-GT, ADA-SCID patients displayed quasi-normal early B cell tolerance. Patients under PEG-ADA showed impaired B cell proliferative responses after BCR/TLR triggering, which normalized in patients treated with HSC-GT. Overall, our data indicate ADA plays an essential role in controlling autoreactive B cell counterselection by regulating BCR and TLR functions. Moreover, our findings confirm the efficacy of HSC-GT in restoring both B-cell tolerance and function. We are currently assessing if B-cell development and function occur properly in ADA-deficiency by studying BM and peripheral blood B-cell development in untreated patients as well as patients following different treatments. Studies are performed in cells from ADA-SCID patients, as well as in the mouse model of ADA-deficiency, before and after treatment with gene therapy, HSC transplant or enzyme replacement therapy.

Our studies will provide crucial information on the role of adenosine metabolism in regulating immune tolerance and its contribution to the pathogenesis of autoimmunity of ADA-SCID in order to improve future therapeutic approaches and patient’s long-term prospects.


Purine metabolite toxicity is believed to be responsible for several organ alterations including neurological/behavioural abnormalities, growth retardation and pulmonary alterations observed in ADA-SCID. However, the precise molecular mechanisms leading to the pathogenesis of the non-immune alterations remain largely unknown. Growth defects and neurological and behavioural alterations have been observed in a significant proportion of ADA-SCID patients, including those surviving after bone marrow transplant or enzyme replacement therapy. We are currently studying the basis of these alterations in the ADA-deficient mouse model. These mice retain many features associated with ADA deficiency in humans, including SCID and a profound metabolic defect. Accumulating ADA substrates lead ADA ko mice to die postnatally within 3 weeks, but mice can be rescued by bone marrow transplant or gene therapy.

Bone and microenvironment defects. We found that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children’s growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling.

Neurological and behavioural alterations. ADA wt and ko littermates are subjected to a number of tests designed to asses learning and behavioural as well as emotional, motivational and exploratory features in young mice. Our preliminary results indicate that ADA ko mice are affected from significant behavioural alterations. We are currently investigating the underlying structural, cellular, and molecular defects in the brain of ADA ko mice, as compared to ADA wt mice.
These studies improve our knowledge on the pathogenesis of the ADA-SCID and contribute to the design of better therapeutic approaches.



The study of haemopoietic stem cells (HSC) dynamics in humans relies currently on data derived from phenotyping, in vitro colony assays or animal models. Different models of hematopoietic hierarchies with “common” and lineage-specific progenitors have been proposed but none of them is supported by direct evidence deriving from studies of in vivo human hematopoiesis. Upon retroviral gene transfer, transduced cells are univocally marked by vector integration sites thus providing a unique tool to track HSC long term in humans by insertional tagging. We previously showed in the context of the ADA-SCID gene therapy that vector transduced CD34+ cells engraft long-term in the absence of aberrant expansion. Our early low-throughput analyses of vector integrations had already revealed the presence of shared vector integrations among different lineages, confirming the engraftment of multilineage HSC (Aiuti et al, 2007). More recently, we were able to show that retroviral insertions are cell-specific and their distribution depends on genetic and epigenetic status of the target cells at the time of transduction (Biasco et al, 2010). The general goal of our project is to study in depth clonal dynamics of human hematopoiesis providing new information on long-term survival, self-renewal, differentiation potential as well as on hierarchical relationship of human HSC, “common” progenitors, and lineage-specific progenitors. To this aim we are generating through the combination of LAM-PCR and pyrosequencing a database of high-throughput vector insertions derived from different retroviral and lentiviral gene therapy clinical trials ongoing and planned at SR-Tiget. Our first approach will be based on studying the effect of disease background and transgene effects on integration site distribution and clonality in vivo in patients at short and long-term in comparison to in vitro integration preference.  We will then validate the activity of single HSC and progenitor cell clones in the patients, identified by the analyses of shared integrants by specific PCR, in vitro differentiation and clonogenic assays and transplant into immunodeficient mice of ex vivo derived HSC. We will also explore the repopulating capacity of distinct human committed progenitors after in vitro transduction and sorting and compare their insertional dataset profile from those retrieved ex vivo from GT patients. Finally, we will establish mathematical models taking in consideration potential biases and addressing hematopoietic system hierarchy and dynamics and challenging the validity of different human hematopoiesis models. To further corroborate our analysis we will compare retroviral tagging with other methods of single cell marking such as vector barcoding, by in vitro and in vivo assay of human hematopoiesis. These studies will provide key information on the survival and multilineage potential maintenance of single, vector transduced HSC clones and will allow to elucidate the composition and structure of haemopoietic hierarchies in humans.


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