Scientific research

Scientific Research


Project Title

Assessing the efficiency of split-inteins mediated protein trans- splicing in correcting the gene defect in ataxia telangiectasia

Principal Investigator

Dr. Giuliana Giardino, MD, PhD- University of Naples “Federico II”

Priority area

Development of Treatments and Therapeutic Interventions

Project Summary

Ataxia-Telangiectasia (A-T, MIM #208900) is a rare recessive disorder caused by mutations in the Ataxia Telangiectasia Mutated (ATM) gene, encoding a serine/threonine protein kinase involved in cell cycle control and repair of DNA double-strand breaks. Although A-T is a multisystemic disorder, the defects in the DNA damage response mainly affect the nervous and immune systems and, to date, it remains an incurable disease. Gene therapy represents an effective strategy to treat patients suffering from A-T, but the large size of ATM coding sequence prevents efficient packaging into vectors commonly used to achieve a stable expression in actively dividing cells, as lentiviral vectors (LVs). Therefore, aim of this project is to exploit the ability of split-inteins-mediated protein-trans-splicing to reconstitute a stable expression of a full-length ATM upon delivery of two different LVs, each expressing a shorter portion of ATM fused to a split-intein.

Project Title

Study on the possible neuro-steroidogenic origin of myelin defects in A-T

Principal Investigator

Flavia Antonucci – University of Milan

Priority area

Aetiology – Understanding the causes of A-T

Project Summary

Children with A-T are characterized by a progressive cerebellar ataxia mainly linked with cerebellar atrophy and atrophy of different myelinated tracts. Demyelination has been reported in cerebella of A-T animal models and humans with A-T; coherently a reduced number of oligodendrocytes (OLs), cells that produce and maintain myelin sheath, has been found in vitro and OLs pathology described in A-T humans. Since neuroactive steroids (NSts) are potent promoters of OLs production/differentiation, we will explore the possible contribution of NSts in A-T aetiology testing the hypothesis that impaired NSts levels in cerebella of male and female ATM-/- embryos lead to OLs pathology during development resulting in the gradual but constant demyelination that, worsening with age, contributes to disease progression. Of note, recent studies showed that NSts has significant therapeutic efficacy in animal models for brain diseases, thus our data may open a new avenue for new approaches in A-T.


Project Title: Regulation of SIRT1 and SIRT7 in human ATM KD and KO cell lines: exploring new targets for therapy

    Principal Investigator: Sabrina Putti (IBBC-CNR)

    Priority area: Prevention of Disease and Conditions, and Promotion of Well-Being – Studies to identify new treatments for A-T

    Project Summary: Sirt1 and Sirt7 are NAD+-dependent protein deacetylases that participate in repairing DNA damage by directly interacting with ATM. Deletion of Sirt7 blocks ATM deacetylation and leads to persistent phosphorylation of ATM, which is critical for DNA repair and cell survival. Sirt7 is associated with nonhomologous end joining (NHEJ) and its deficiency inhibits NHEJ efficiency. Sirt1 is also involved in DNA repair and is recruited to breaks in an ATM-dependent manner. Sirt1 is found to be involved in the regulation of homologous recombination (HR) and NHEJ repair pathways. Mutated forms of ATM with compromised kinase activity, kinase dead (ATM KD , lead to a more severe impairment of the homologous recombination process than the absence of the protein (ATM KO). Therefore, we hypothesize a direct relationship between ATM KD and the deacetylases Sirt1 and Sirt7, and we aim to analyze their expression in cells isolated from ATM-KD and KO mutant mice, as well as in human ATM kinase-inactive or KO cell lines. By increasing the level and activity of these deacetylases, we will attempt to improve DNA damage repair response and chromosomal stability, because they regulate the DDR by modulating also other targets then ATM.

    Project Title:  Toward a personalized gene therapy in A-T: the role of 4-53 and synthetic ATM variants.

    Principal Investigator: Prof. Michele Menotta, University of Urbino

    Priority area: Prevention of Disease and Conditions, and Promotion of Well-Being – Studies to identify new treatments for A-T

    Project Summary: Our research team has been working for some years to understand the mechanisms of action of dexamethasone in A-T pathology since the Urbino operating unit has been involved in the Erydex clinical trials. During the investigations it emerged that there are other ATM forms that are much smaller than native ATM but weakly expressed in cells. We have recently characterized some of these ATM variants from a biochemical point of view (miniATM, ATM 3-52, ATM 4-53 and ATM SINT) and it has emerged that some of them complemented the A-T phenotype. The aim of the project is to verify whether the functional complementation of the ATM variants (ATM 4-53 and ATM SINT in this case) depends on the residual endogenous ATM (albeit mutated) and therefore the complementation could be a genotype dependent event. The final scope is to use the small ATM variants for a potential delivery therapy using AAV vectors, the only ones currently approved and utilized in gene therapy, or by nanocarriers.


    Ataxia Telangiectasia: a new murine model to discover the connection between Purkinje cell calcium homeostasis disruption and disease pathogenesis
    Prof. Eriola Hoxha. Department of Neuroscience/ University of Turin/ Neuroscience
    Institute Cavalieri Ottolenghi (NICO)/ Laboratory of Neurophysiology of Neurodegenerative Diseases.

    Priority area: Aetiology-Understanding the causes of A-T
    Project Summary:
    The progressive cerebellar ataxia in A-T is due to the loss of cerebellar granule and Purkinje cells (PCs). The mechanisms and the reason of the cerebellar degeneration are still not clear. Furthermore, it is not clear how the cerebellar circuitry is modified during the progression of the A-T disease. Recently, we generated a new murine model of A-T with a catalytically inactive Atm called “kinase dead”. By exploiting this mouse model, our proposal is aimed at finding the dysfunctions of cerebellar PCs in an early symptomatic stage. More specifically, PCs of an A-T mouse model with motor deficits will be studied to unveil dysfunctions in glutamatergic transmission and calcium signaling.
    We expect that our work will provide, for the first time, a comprehensive study of signal processing in the
    cerebellar circuitry of a symptomatic A-T model. Unraveling the functional mechanisms that occur in the
    cerebellum during A-T is a promising way to provide clues for ataxia treatment in A-T.

    Fluoxetine reveres cognitive defects in a mouse model of AT: a preclinical study to elucidate the precise mechanism of action.
    Prof. Flavia Antonucci – University of Milan
    Priority area: Evaluation of Treatments and Therapeutic Interventions – Studies to prove how new treatments work and understand how to use them
    Project Summary:
    The neurodegenerative condition associated to AT presents impaired cognitive abilities largely described but poorly investigated. We recently displayed that reduced levels of ATM as well as the inhibition of its kinase activity generate developmental changes and enhanced inhibition in the hippocampus. Coherently with these findings, we found impaired cognitive behaviours in AT heterozygous and KO mice that we managed to recover by two different in vivo protocols based on the systemic administrations of antidepressant Fluoxetine (Fluox). Thus, in this project, we aim at unveiling how Fluoxetine reverses defective cognitive behaviours and to this purpose we will use an array of biochemical and electrophysiological approaches. This project will lay scientific basis for a safe and conscious administration of Fluox in AT individuals in the attempt to correct cognitive defects.


    New perspectives in E-Care: Exploring virtual in-home multiple interventions for Ataxia Telangiectasia (Acronym: NEW_EXIT)

    Prof. Agata Polizzi, Department of Educational Science, University of Catania, Catania

    Priority area: Management of Diseases and Conditions

      Various areas of Neurology have been addressed to the use of telemedicine, however many rare neurological diseases still lack such opportunity. Care of Ataxia Telangiectasia (AT) patients is entrusted to tailored supports and rehabilitative programs. Difficulties in accessing specialized care prompt the need of alternative strategies. We aim to virtually in-home explore motion, motor imagery ability, cognition and affect in AT patients to deep the knowledge on these investigated areas and experiment a model of e-care to ameliorate AT quality of life.

      Zebrafish disease-model of Ataxia Telangiectasia: a tool to identify potential new therapeutic opportunities

      Dr. Chiara Gabellini, Department of Biology, University of Pisa, Unit of Cellular and Developmental Biology Priority area: Underpinning Research

      The protein kinase ATM is a master regulator of the genotoxic stress response, however, the defective defence against DNA-damage may not be the unique responsible for neurodegeneration in AT patients. Since ATM protein maintains the redox homeostasis through autophagy modulation, we will investigate the role of this process in AT pathogenesis through the characterization of a mutant ATM zebrafish line. This disease-model may support the identification of new targeted therapies for AT treatment.

      p53 Mitotic Centrosome Localization as a functional test to predict pathogenicity of ATM missense variants

      Dr. Giulia Federici, IRCCS Regina Elena National Cancer Institute, UOSD of Cellular Networks and Molecular Therapeutic Targets

      Priority area: Detection, Screening and Diagnosis

      The ATM gene, whose mutation is responsible for the Ataxia-Telangiectasia (A-T), can be affected by thousands of different mutational events. The best-known mutations are the nonsense truncating mutations, which result in ATM protein disruption and cause A-T (the so-called pathogenic variants). Less well-characterized but more frequent are the missense variants which do not directly disrupt the protein, induce less evident effects on ATM function/stability, and their disease contribution (pathogenicity) is unclear. This poses substantial limitation to the clinical impact of sequence information. We propose to genetically validate the possibility of predicting the pathogenicity of ATM missense variants by employing a novel ATM functional test we recently developed. The test measures ATM-dependent localization of p53 at the centrosomes during mitosis (p53-MCL) and recognizes ATM pathogenic variants. Here, we will generate a collection of cell clones with a series of ATM missense variants by CRISPR/Cas9- induced genome editing and used them to validate the p53-MCL predictions.


      Comparison of immunologic, biochemical, and genomic profile between steroid-responsive and non-steroid-responsive patients with A-T

      Dr. Emilia Cirillo, MD, PhD, Pediatrician in service at the Level I Program in Pediatric Immunology directed by Prof. Claudio Pignata at the Maternal-Children’s Department of the AOU Policlinico Federico II (Naples, Italy)

      5-Development of Treatments and Therapeutic Interventions

        Several studies have documented a beneficial effect of betamethasone, a drug of the steroid family, on neurological and immunological symptoms of patients with Ataxia-Telangiectasia. However, some patients respond to the treatment while others do not benefit. The aim of this project is to study in vitro the functions and some biochemical mechanisms of lymphocytes of patients with Ataxia-Telangiectasia, in order to better understand the variability of response to betamethasone. Specifically, the study aims to assess whether variability in neurological response to steroids is similar to lymphocyte behavior. Moreover, the use of new “omics” technologies will allow a better understanding of the molecular mechanisms underlying the different effect of cortisone on peripheral lymphocytes.

        Modulation of autophagy as a potential therapeutic approach for Ataxia Telangiectasia

        Dr. Venturina Stagni, PhD, Institute of Molecular Biology and Pathology, CNR Via Degli Apuli 4 (Rome)

        5-Development of Treatments and Therapeutic Interventions

        The future development of molecularly targeted therapies for patients with Ataxia-Telangiectasia (AT) is highly dependent on further understanding of the cellular signaling pathways regulated by ATM kinase. Autophagy is a highly deregulated cellular signaling pathway in neurodegenerative diseases and reactivation of normal function of this pathway appears to strongly alleviate the neurodegenerative phenotype in several diseases. The aim of this project is to study the role of regulation of the autophagy pathway by ATM in the pathogenesis of AT, based on recent literature that identifies a role of this kinase in the modulation of this cellular signaling pathway in response to oxidative stress. In addition, a new AT model will be developed in the Zebrafish (D. rerio) model organism that will allow future drug screening experiments for AT in vivo.

        Do deficits in cerebellar circuitry precede neurodegeneration in Ataxia Telangiectasia?

        Dr. Eriola Hoxha PhD, Cavalieri Ottolenghi Institute of Neuroscience, Regione Gonzole, 10 – 10043 Orbassano (Torino)


        The discovery of factors related to cerebellar ataxia in a mouse model of A-T, at an early stage of the disease, highlights the importance of early intervention. We aim to find functional cerebellar deficits that precede degeneration, in the mouse model, responsible for ataxia. We will focus on finding alterations in electrical signals, underlying cerebellar function. We will provide information to address cell death that is a cause of ataxia.


        Gene therapy in A-T

        Dr. Manuela Pellegrini (IBCN-CNR, Monterotondo, RM)

        5-Development of Treatments and Therapeutic Interventions

          Ataxia Telangiectasia (A-T) is a rare genetic disease caused by mutations in the ATM gene. The aim of the project is to insert a corrected version of the ATM gene by a lentiviral strategy in human cells of A-T patients and in hematopoietic progenitor cells of mouse models lacking the protein. Recovering ATM activity is expected to reverse the hematopoietic phenotypes of the disease, paving the way for gene therapy in A-T patients.

          Glutamine metabolism in A-T cells

          Dr. Michele Menotta (Dept. of Biomolecular Sciences, University of Urbino)

          6-Evaluation of treatments and therapeutic interventions

          Recent studies have hypothesized a possible role of glutamine in the pathophysiology of the disease and a possible effect of dexamethasone at the metabolic level. The present project aims to investigate the role of glutamine metabolism in AT cells, at basal level and in response to the drug. The results will allow proposing a dexamethasone-based therapy to treat neurological and metabolic symptoms.

          Corticosteroids for the treatment of neurological symptoms

          The evidence that proved now that the AT cells are in a continuous state of oxidative stress suggested the possibility of treating the deficiency of ATM with exogenous contribution of antioxidants: the experimental studies are in progress. The idea to use the corticosteroids such as Betamethasone started in 2006 beginning with the empirical observation of the impressive neurological improvement presented by a girl with AT of six years old treated with betamethasone in order to cure un episode of asthma. Since then, there have been carried out different clinical trials at the major centers which deal with AT, proving that most of the treated patients respond very positively at betamethasone. However, the drug’s effect disappears quickly after stopping the administration. Therefore, in the study there are still new methods of betamethasone consumption in order to arrive to a continuous treatment with low dose of corticosteroids.

          Intra-erythrocyte Dexamethasone

          To avoid the toxicity of long treatments with oral betamethasone, in 2010 it was proposed to use the encapsulated dexamethasone in the erythrocytes for the slow and sustained release of n circle corticosteroids. The first clinical trial carried out on 22 Italian patients ended successfully and the results were published at the beginning of 2014. Till present a Worldwide Clinical Study of 3rd Phase for the registration of intra-erythrocyte dexamethasone for the treatment of neurological symptoms in AT is in progress. At the present time the study is in enrolment phase and will provide the treatment of 180 patients in more than 20 countries of the world besides in Italy (for further informations

          Research projects financed by ANAT in Italy.

          Different excellence centers in Italy and in the world are involved in research projects aimed to define the role of ATM protein in the AT physiopathology the ATM protein. To know the mechanisms that lead to neurodegeneration as a result of the lack of ATM and basilar protein to understand the symptoms of the patients with AT and to propose new therapeutic strategies. ANAT finances for years the following research projects:

          In the last years it was proposed that ATM protein is involved in defense mechanisms to oxidative stress and in metabolism.

          Project’s title: The role of ATM in the control of cellular metabolism

          Scientific responsible: Doc. Vincenzo Costanzo

          Host institute: IFOM, FIRC Institute of Molecular Oncology (Milan)

          The molecular basis of corticosteroids efficiency in the improvement of neurological symptoms at patients with AT are still research objects.

          Project title: Corticosteroids for patients with Ataxia Telangiectasia (AT): the dexamethasone effect on AT cells activity

          Scientific responsibles: Doc. Alfredo Brusco, Doc. Simona Cavalieri

          Host institute: Department of Medical Sciences, University of Turin

          Very interesting is the observation that dexamethasone, similar to betamethasone, behaves modulating the G6PD enzyme activity and, therefore, regulating the cellular oxidative stress.

          Project title: Molecular defects associated to ATM genes and the possible therapeutical approach

          Scientific responsible: Prof. Mauro Magnani

          Host institute: Department of Bimolecular Sciences, University of Urbino