Publications by authors named "Immacolata Brigida"

31 Publications

Lentiviral correction of enzymatic activity restrains macrophage inflammation in adenosine deaminase 2 deficiency.

Blood Adv 2021 08;5(16):3174-3187

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Adenosine deaminase 2 deficiency (DADA2) is a rare inherited disorder that is caused by autosomal recessive mutations in the ADA2 gene. Clinical manifestations include early-onset lacunar strokes, vasculitis/vasculopathy, systemic inflammation, immunodeficiency, and hematologic defects. Anti-tumor necrosis factor therapy reduces strokes and systemic inflammation. Allogeneic hematopoietic stem/progenitor cell (HSPC) transplantation can ameliorate most disease manifestations, but patients are at risk for complications. Autologous HSPC gene therapy may be an alternative curative option for patients with DADA2. We designed a lentiviral vector encoding ADA2 (LV-ADA2) to genetically correct HSPCs. Lentiviral transduction allowed efficient delivery of the functional ADA2 enzyme into HSPCs from healthy donors. Supranormal ADA2 expression in human and mouse HSPCs did not affect their multipotency and engraftment potential in vivo. The LV-ADA2 induced stable ADA2 expression and corrected the enzymatic defect in HSPCs derived from DADA2 patients. Patients' HSPCs re-expressing ADA2 retained their potential to differentiate into erythroid and myeloid cells. Delivery of ADA2 enzymatic activity in patients' macrophages led to a complete rescue of the exaggerated inflammatory cytokine production. Our data indicate that HSPCs ectopically expressing ADA2 retain their multipotent differentiation ability, leading to functional correction of macrophage defects. Altogether, these findings support the implementation of HSPC gene therapy for DADA2.
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http://dx.doi.org/10.1182/bloodadvances.2020003811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8405196PMC
August 2021

A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.

J Exp Med 2019 12 10;216(12):2778-2799. Epub 2019 Oct 10.

Baylor-Hopkins Center for Mendelian Genomics, Houston, TX.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.
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http://dx.doi.org/10.1084/jem.20190147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888978PMC
December 2019

Targeted NGS Platforms for Genetic Screening and Gene Discovery in Primary Immunodeficiencies.

Front Immunol 2019 11;10:316. Epub 2019 Apr 11.

Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy.

Primary Immunodeficiencies (PIDs) are a heterogeneous group of genetic immune disorders. While some PIDs can manifest with more than one phenotype, signs, and symptoms of various PIDs overlap considerably. Recently, novel defects in immune-related genes and additional variants in previously reported genes responsible for PIDs have been successfully identified by Next Generation Sequencing (NGS), allowing the recognition of a broad spectrum of disorders. To evaluate the strength and weakness of targeted NGS sequencing using custom-made Ion Torrent and Haloplex (Agilent) panels for diagnostics and research purposes. Five different panels including known and candidate genes were used to screen 105 patients with distinct PID features divided in three main PID categories: and . The Ion Torrent sequencing platform was used in 73 patients. Among these, 18 selected patients without a molecular diagnosis and 32 additional patients were analyzed by Haloplex enrichment technology. The complementary use of the two custom-made targeted sequencing approaches allowed the identification of causative variants in 28.6% ( = 30) of patients. Twenty-two out of 73 (34.6%) patients were diagnosed by Ion Torrent. In this group 20 were included in the SCID/CID category. Eight out of 50 (16%) patients were diagnosed by Haloplex workflow. Ion Torrent method was highly successful for those cases with well-defined phenotypes for immunological and clinical presentation. The Haloplex approach was able to diagnose 4 SCID/CID patients and 4 additional patients with complex and extended phenotypes, embracing all three PID categories in which this approach was more efficient. Both technologies showed good gene coverage. NGS technology represents a powerful approach in the complex field of rare disorders but its different application should be weighted. A relatively small NGS target panel can be successfully applied for a robust diagnostic suspicion, while when the spectrum of clinical phenotypes overlaps more than one PID an in-depth NGS analysis is required, including also whole exome/genome sequencing to identify the causative gene.
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http://dx.doi.org/10.3389/fimmu.2019.00316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470723PMC
September 2020

Lentiviral haemopoietic stem/progenitor cell gene therapy for treatment of Wiskott-Aldrich syndrome: interim results of a non-randomised, open-label, phase 1/2 clinical study.

Lancet Haematol 2019 May 10;6(5):e239-e253. Epub 2019 Apr 10.

CSD Pharma Consulting, Redhill, UK; Orchard Therapeutics, London, UK.

Background: Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy.

Methods: We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32).

Findings: Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1-12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5-5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8-35·6) before gene therapy to 66·7% (55·7-98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1-31·0) to 76·6% (53·1-98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44-3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04-1·11) per PYO in the second year after gene therapy and 0·17 (0·00-0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 10 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 × 10 per L in one patient, 50-100 × 10 per L in five patients, and more than 100 × 10 per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy.

Interpretation: Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available.

Funding: Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.
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http://dx.doi.org/10.1016/S2352-3026(19)30021-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494976PMC
May 2019

First Case of Patient With Two Homozygous Mutations in and Genes Presenting With Pyogenic Bacterial Infections, Elevated IgE, and Persistent EBV Viremia.

Front Immunol 2019 14;10:130. Epub 2019 Feb 14.

University Department of Pediatrics, Unit of Immune and Infectious Diseases, Childrens' Hospital Bambino Gesù, Rome, Italy.

We described for the first time a female patient with the simultaneous presence of two homozygous mutations in and genes presenting with pyogenic bacterial infections, elevated IgE, and persistent EBV viremia. In addition to defective TLR/IL1R-signaling, we described novel functional alterations into the myeloid compartment. In particular, we demonstrated a defective production of reactive oxygen species exclusively in monocytes upon stimulation, the inability of immature mono-derived DCs (iDCs) to differentiate into mature DCs (mDCs) and the incapacity of mono-derived macrophages (MDMs) to resolve BCG infection . Our data do not provide any evidence for digenic inheritance in our patient, but rather for the association of two monogenic disorders. This case illustrates the importance of using next generation sequencing (NGS) to determine the most accurate and early diagnosis in atypical clinical and immunological phenotypes, and with particular concern in consanguineous families. Indeed, besides the increased susceptibility to recurrent invasive pyogenic bacterial infections due to MYD88 deficiency, the identification of mutations underline the risk of developing invasive fungal infections emphasizing the careful monitoring for the occurrence of fungal infection and the opportunity of long-term antifungal prophylaxis.
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http://dx.doi.org/10.3389/fimmu.2019.00130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383679PMC
December 2019

ALPS-Like Phenotype Caused by ADA2 Deficiency Rescued by Allogeneic Hematopoietic Stem Cell Transplantation.

Front Immunol 2018 14;9:2767. Epub 2019 Jan 14.

Paediatric Hematology-Oncology, Ospedale della Donna e del Bambino, Verona, Italy.

Adenosine deaminase 2 (ADA2) deficiency is an auto-inflammatory disease due to mutations in cat eye syndrome chromosome region candidate 1 () gene, currently named . The disease has a wide clinical spectrum encompassing early-onset vasculopathy (targeting skin, gut and central nervous system), recurrent fever, immunodeficiency and bone marrow dysfunction. Different therapeutic options have been proposed in literature, but only steroids and anti-cytokine monoclonal antibodies (such as tumor necrosis factor inhibitor) proved to be effective. If a suitable donor is available, hematopoietic stem cell transplantation (HSCT) could be curative. Here we describe a case of ADA2 deficiency in a 4-year-old Caucasian girl. The patient was initially classified as autoimmune neutropenia and then she evolved toward an autoimmune lymphoproliferative syndrome (ALPS)-like phenotype. The diagnosis of ALPS became uncertain due to atypical clinical features and normal FAS-induced apoptosis test. She was treated with G-CSF first and subsequently with immunosuppressive drugs without improvement. Only HSCT from a 9/10 HLA-matched unrelated donor, following myeloablative conditioning, completely solved the clinical signs related to ADA2 deficiency. Early diagnosis in cases presenting with hematological manifestations, rather than classical vasculopathy, allows the patients to promptly undergo HSCT and avoid more severe evolution. Finally, in similar cases highly suspicious for genetic disease, it is desirable to obtain molecular diagnosis before performing HSCT, since it can influence the transplant procedure. However, if HSCT has to be performed without delay for clinical indication, related donors should be excluded to avoid the risk of relapse or partial benefit due to a hereditary genetic defect.
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http://dx.doi.org/10.3389/fimmu.2018.02767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339927PMC
October 2019

T-cell defects in patients with germline mutations account for combined immunodeficiency.

Blood 2018 11 25;132(22):2362-2374. Epub 2018 Sep 25.

Department of Pediatrics & Department of Microbiology-Immunology, Dalhousie University, Halifax, NS, Canada.

ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper-immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α-directed migration. Gene transfer of in patients' T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8 T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.
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http://dx.doi.org/10.1182/blood-2018-07-863431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265646PMC
November 2018

Next-Generation Sequencing Reveals A JAGN1 Mutation in a Syndromic Child With Intermittent Neutropenia.

J Pediatr Hematol Oncol 2019 05;41(4):e266-e269

University Department of Pediatrics, Unit of Immune and Infectious Diseases, Childrens' Hospital Bambino Gesù.

Background: Jagunal homolog 1 (JAGN1) gene was identified as a novel responsible for severe congenital neutropenia. The protein encoded by this gene is required for neutrophil differentiation, survival and function in microbial activity. JAGN1-deficient human neutrophils are characterized by alterations in trafficking within the endoplasmic reticulum and golgi compartments because of ultrastructural defects in endoplasmic reticulum and susceptibility to apoptosis.

Observations: We report a patient exhibiting an intermittent neutropenia, for which a next-generation sequencing revealed a homozygous mutation in the JAGN1 gene.

Conclusions: The patient extends the clinical variability associated to JAGN1 mutations, and this case highlights the importance of genetic investigations in patients with suspected neutropenia.
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http://dx.doi.org/10.1097/MPH.0000000000001256DOI Listing
May 2019

First Occurrence of Plasmablastic Lymphoma in Adenosine Deaminase-Deficient Severe Combined Immunodeficiency Disease Patient and Review of the Literature.

Front Immunol 2018 2;9:113. Epub 2018 Feb 2.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Pediatric Immunohematology and Bone Marrow Transplantation Unit, Scientific Institute San Raffaele (IRCCS), Milan, Italy.

Adenosine deaminase-deficient severe combined immunodeficiency disease (ADA-SCID) is a primary immune deficiency characterized by mutations in the ADA gene resulting in accumulation of toxic compounds affecting multiple districts. Hematopoietic stem cell transplantation (HSCT) from a matched donor and hematopoietic stem cell gene therapy are the preferred options for definitive treatment. Enzyme replacement therapy (ERT) is used to manage the disease in the short term, while a decreased efficacy is reported in the medium-long term. To date, eight cases of lymphomas have been described in ADA-SCID patients. Here we report the first case of plasmablastic lymphoma occurring in a young adult with ADA-SCID on long-term ERT, which turned out to be Epstein-Barr virus associated. The patient previously received infusions of genetically modified T cells. A cumulative analysis of the eight published cases of lymphoma from 1992 to date, and the case here described, reveals a high mortality (89%). The most common form is diffuse large B-cell lymphoma, which predominantly occurs in extra nodal sites. Seven cases occurred in patients on ERT and two after haploidentical HSCT. The significant incidence of immunodeficiency-associated lymphoproliferative disorders and poor survival of patients developing this complication highlight the priority in finding a prompt curative treatment for ADA-SCID.
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http://dx.doi.org/10.3389/fimmu.2018.00113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801298PMC
March 2019

Biological and functional characterization of bone marrow-derived mesenchymal stromal cells from patients affected by primary immunodeficiency.

Sci Rep 2017 08 15;7(1):8153. Epub 2017 Aug 15.

Department of Pediatric Hematology/Oncology, IRCCS Bambino Gesù Children's Hospital, Rome, Italy.

Mesenchymal stromal cells (MSCs) represent a key component of bone marrow (BM) microenvironment and display immune-regulatory properties. We performed a detailed analysis of biological/functional properties of BM-MSCs derived from 33 pediatric patients affected by primary immune-deficiencies (PID-MSCs): 7 Chronic Granulomatous Disease (CGD), 15 Wiskott-Aldrich Syndrome (WAS), 11 Severe Combined Immunodeficiency (SCID). Results were compared with MSCs from 15 age-matched pediatric healthy-donors (HD-MSCs). Clonogenic and proliferative capacity, differentiation ability, immunophenotype, immunomodulatory properties were analyzed. WB and RT-qPCR for CYBB, WAS and ADA genes were performed. All PID-MSCs displayed clonogenic and proliferative capacity, morphology and immunophenotype comparable with HD-MSCs. PID-MSCs maintained the inhibitory effect on T- and B-lymphocyte proliferation, except for decreased inhibitory ability of SCID-MSCs at MSC:PBMC ratio 1:10. While HD- and CGD-MSCs were able to inhibit monocyte maturation into immature dendritic cells, in SCID- and WAS-MSCs this ability was reduced. After Toll-like Receptor priming, PID-MSCs displayed in vitro an altered gene expression profile of pro- and anti-inflammatory soluble factors. PID-MSCs displayed lower PPARγ levels and WAS- and SCID-MSCs higher levels of key osteogenic markers, as compared with HD-MSCs. Our results indicate that PID-MSCs may be defective in some functional abilities; whether these defects contribute to disease pathophysiology deserves further investigation.
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http://dx.doi.org/10.1038/s41598-017-08550-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557950PMC
August 2017

Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency.

Blood 2016 07 29;128(1):45-54. Epub 2016 Apr 29.

San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy; Pediatric Immunohematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy;

Adenosine deaminase (ADA) deficiency is a rare, autosomal-recessive systemic metabolic disease characterized by severe combined immunodeficiency (SCID). The treatment of choice for ADA-deficient SCID (ADA-SCID) is hematopoietic stem cell transplant from an HLA-matched sibling donor, although <25% of patients have such a donor available. Enzyme replacement therapy (ERT) partially and temporarily relieves immunodeficiency. We investigated the medium-term outcome of gene therapy (GT) in 18 patients with ADA-SCID for whom an HLA-identical family donor was not available; most were not responding well to ERT. Patients were treated with an autologous CD34(+)-enriched cell fraction that contained CD34(+) cells transduced with a retroviral vector encoding the human ADA complementary DNA sequence (GSK2696273) as part of single-arm, open-label studies or compassionate use programs. Overall survival was 100% over 2.3 to 13.4 years (median, 6.9 years). Gene-modified cells were stably present in multiple lineages throughout follow up. GT resulted in a sustained reduction in the severe infection rate from 1.17 events per person-year to 0.17 events per person-year (n = 17, patient 1 data not available). Immune reconstitution was demonstrated by normalization of T-cell subsets (CD3(+), CD4(+), and CD8(+)), evidence of thymopoiesis, and sustained T-cell proliferative capacity. B-cell function was evidenced by immunoglobulin production, decreased intravenous immunoglobulin use, and antibody response after vaccination. All 18 patients reported infections as adverse events; infections of respiratory and gastrointestinal tracts were reported most frequently. No events indicative of leukemic transformation were reported. Trial details were registered at www.clinicaltrials.gov as #NCT00598481.
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http://dx.doi.org/10.1182/blood-2016-01-688226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5325048PMC
July 2016

A novel genomic inversion in Wiskott-Aldrich-associated autoinflammation.

J Allergy Clin Immunol 2016 08 22;138(2):619-622.e7. Epub 2016 Apr 22.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy; Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.

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http://dx.doi.org/10.1016/j.jaci.2016.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4969072PMC
August 2016

The case of an APDS patient: Defects in maturation and function and decreased in vitro anti-mycobacterial activity in the myeloid compartment.

Clin Immunol 2017 05 28;178:20-28. Epub 2015 Dec 28.

University Department of Pediatrics, Unit of Immune and Infectious Diseases, Childrens' Hospital Bambino Gesù, Rome, Italy. Electronic address:

Activated PI3-kinase delta syndrome (APDS) was recently reported as a novel primary immunodeficiency caused by heterozygous gain-of-function mutations in PIK3CD gene. Here we describe immunological studies in a 19year old APDS patient for whom genetic diagnosis was discovered by Whole Exome Sequencing (WES) analysis. In addition to the progressive lymphopenia and defective antibody production we showed that the ability of the patient's B cells to differentiate in vitro is severely reduced. An in depth analysis of the myeloid compartment showed an increased expression of CD83 activation marker on monocytes and mono-derived DC cells. Moreover, monocytes-derived macrophages (MDMs) failed to solve the Mycobacterium bovis bacillus Calmette Guèrin (BCG) infection in vitro. Selective p110δ inhibitor IC87114 restored the MDM capacity to kill BCG in vitro. Our data show that the constitutive activation of Akt-mTOR pathway induces important alterations also in the myeloid compartment providing new insights in order to improve the therapeutic approach in these patients.
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http://dx.doi.org/10.1016/j.clim.2015.12.008DOI Listing
May 2017

B-cell reconstitution after lentiviral vector-mediated gene therapy in patients with Wiskott-Aldrich syndrome.

J Allergy Clin Immunol 2015 Sep 16;136(3):692-702.e2. Epub 2015 Mar 16.

San Raffaele Telethon Institute for Gene Therapy (TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy.

Background: Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however, administration of WAS gene-corrected autologous hematopoietic stem cells has been demonstrated as a feasible alternative therapeutic approach.

Objective: Because B-cell homeostasis is perturbed in patients with WAS and restoration of immune competence is one of the main therapeutic goals, we have evaluated reconstitution of the B-cell compartment in 4 patients who received autologous hematopoietic stem cells transduced with lentiviral vector after a reduced-intensity conditioning regimen combined with anti-CD20 administration.

Methods: We evaluated B-cell counts, B-cell subset distribution, B cell-activating factor and immunoglobulin levels, and autoantibody production before and after gene therapy (GT). WAS gene transfer in B cells was assessed by measuring vector copy numbers and expression of Wiskott-Aldrich syndrome protein.

Results: After lentiviral vector-mediated GT, the number of transduced B cells progressively increased in the peripheral blood of all patients. Lentiviral vector-transduced progenitor cells were able to repopulate the B-cell compartment with a normal distribution of B-cell subsets both in bone marrow and the periphery, showing a WAS protein expression profile similar to that of healthy donors. In addition, after GT, we observed a normalized frequency of autoimmune-associated CD19(+)CD21(-)CD35(-) and CD21(low) B cells and a reduction in B cell-activating factor levels. Immunoglobulin serum levels and autoantibody production improved in all treated patients.

Conclusions: We provide evidence that lentiviral vector-mediated GT induces transgene expression in the B-cell compartment, resulting in ameliorated B-cell development and functionality and contributing to immunologic improvement in patients with WAS.
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http://dx.doi.org/10.1016/j.jaci.2015.01.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4559137PMC
September 2015

B-cell development and functions and therapeutic options in adenosine deaminase-deficient patients.

J Allergy Clin Immunol 2014 Mar 5;133(3):799-806.e10. Epub 2014 Feb 5.

San Raffaele Telethon Institute for Gene Therapy (TIGET), San Raffaele Scientific Institute, Milan, Italy; Department of Systems Medicine, Tor Vergata University, Rome, Italy. Electronic address:

Background: Adenosine deaminase (ADA) deficiency causes severe cellular and humoral immune defects and dysregulation because of metabolic toxicity. Alterations in B-cell development and function have been poorly studied. Enzyme replacement therapy (ERT) and hematopoietic stem cell (HSC) gene therapy (GT) are therapeutic options for patients lacking a suitable bone marrow (BM) transplant donor.

Objective: We sought to study alterations in B-cell development in ADA-deficient patients and investigate the ability of ERT and HSC-GT to restore normal B-cell differentiation and function.

Methods: Flow cytometry was used to characterize B-cell development in BM and the periphery. The percentage of gene-corrected B cells was measured by using quantitative PCR. B cells were assessed for their capacity to proliferate and release IgM after stimulation.

Results: Despite the severe peripheral B-cell lymphopenia, patients with ADA-deficient severe combined immunodeficiency showed a partial block in central BM development. Treatment with ERT or HSC-GT reverted most BM alterations, but ERT led to immature B-cell expansion. In the periphery transitional B cells accumulated under ERT, and the defect in maturation persisted long-term. HSC-GT led to a progressive improvement in B-cell numbers and development, along with increased levels of gene correction. The strongest selective advantage for ADA-transduced cells occurred at the transition from immature to naive cells. B-cell proliferative responses and differentiation to immunoglobulin secreting IgM after B-cell receptor and Toll-like receptor triggering were severely impaired after ERT and improved significantly after HSC-GT.

Conclusions: ADA-deficient patients show specific defects in B-cell development and functions that are differently corrected after ERT and HSC-GT.
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http://dx.doi.org/10.1016/j.jaci.2013.12.1043DOI Listing
March 2014

Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity.

Clin Immunol 2013 Mar 24;146(3):248-61. Epub 2013 Jan 24.

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.

Cell-surface CD25 expression is critical for maintaining immune function and homeostasis. As in few reported cases, CD25 deficiency manifests with severe autoimmune enteritis and viral infections. To dissect the underlying immunological mechanisms driving these symptoms, we analyzed the regulatory and effector T cell functions in a CD25 deficient patient harboring a novel IL2RA mutation. Pronounced lymphoproliferation, mainly of the CD8(+) T cells, was detected together with an increase in T cell activation markers and elevated serum cytokines. However, Ag-specific responses were impaired in vivo and in vitro. Activated CD8(+)STAT5(+) T cells with lytic potential infiltrated the skin, even though FOXP3(+) Tregs were present and maintained a higher capacity to respond to IL-2 compared to other T-cell subsets. Thus, the complex pathogenesis of CD25 deficiency provides invaluable insight into the role of IL2/IL-2RA-dependent regulation in autoimmunity and inflammatory diseases.
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http://dx.doi.org/10.1016/j.clim.2013.01.004DOI Listing
March 2013

Autoimmune dysregulation and purine metabolism in adenosine deaminase deficiency.

Front Immunol 2012 27;3:265. Epub 2012 Aug 27.

San Raffaele Telethon Institute for Gene Therapy Milan, Italy.

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, severely impaired cellular and humoral immunity, failure to thrive, and recurrent infections. 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 (HSC-GT). Although varying degrees of immune reconstitution can be achieved by these treatments, breakdown of tolerance is a major concern in ADA-SCID. Immune dysregulation such as autoimmune hypothyroidism, diabetes mellitus, hemolytic anemia, and immune thrombocytopenia are frequently observed in milder forms of the disease. However, several reports document similar complications also in patients on long-term PEG-ADA and after BMT or GT treatment. A skewed repertoire and decreased immune functions have been implicated in autoimmunity observed in certain B-cell and/or T-cell immunodeficiencies, but it remains unclear to what extent specific mechanisms of tolerance are affected in ADA deficiency. Herein we provide an overview about ADA-SCID and the autoimmune manifestations reported in these patients before and after treatment. We also assess the value of the ADA-deficient mouse model as a useful tool to study both immune and metabolic disease mechanisms. With focus on regulatory T- and B-cells we discuss the lymphocyte subpopulations particularly prone to contribute to the loss of self-tolerance and onset of autoimmunity in ADA deficiency. Moreover we address which aspects of immune dysregulation are specifically related to alterations in purine metabolism caused by the lack of ADA and the subsequent accumulation of metabolites with immunomodulatory properties.
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http://dx.doi.org/10.3389/fimmu.2012.00265DOI Listing
September 2012

Autoimmune dysregulation and purine metabolism in adenosine deaminase deficiency.

Front Immunol 2012 27;3:265. Epub 2012 Aug 27.

San Raffaele Telethon Institute for Gene Therapy Milan, Italy.

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, severely impaired cellular and humoral immunity, failure to thrive, and recurrent infections. 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 (HSC-GT). Although varying degrees of immune reconstitution can be achieved by these treatments, breakdown of tolerance is a major concern in ADA-SCID. Immune dysregulation such as autoimmune hypothyroidism, diabetes mellitus, hemolytic anemia, and immune thrombocytopenia are frequently observed in milder forms of the disease. However, several reports document similar complications also in patients on long-term PEG-ADA and after BMT or GT treatment. A skewed repertoire and decreased immune functions have been implicated in autoimmunity observed in certain B-cell and/or T-cell immunodeficiencies, but it remains unclear to what extent specific mechanisms of tolerance are affected in ADA deficiency. Herein we provide an overview about ADA-SCID and the autoimmune manifestations reported in these patients before and after treatment. We also assess the value of the ADA-deficient mouse model as a useful tool to study both immune and metabolic disease mechanisms. With focus on regulatory T- and B-cells we discuss the lymphocyte subpopulations particularly prone to contribute to the loss of self-tolerance and onset of autoimmunity in ADA deficiency. Moreover we address which aspects of immune dysregulation are specifically related to alterations in purine metabolism caused by the lack of ADA and the subsequent accumulation of metabolites with immunomodulatory properties.
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http://dx.doi.org/10.3389/fimmu.2012.00265DOI Listing
September 2012

Defective B cell tolerance in adenosine deaminase deficiency is corrected by gene therapy.

J Clin Invest 2012 Jun 24;122(6):2141-52. Epub 2012 May 24.

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy.

Adenosine deaminase (ADA) gene defects are among the most common causes of SCID. Restoration of purine metabolism and immune functions can be achieved by enzyme replacement therapy, or more effectively by bone marrow transplant or HSC gene therapy (HSC-GT). However, autoimmune complications and autoantibody production, including anti-nuclear antibodies (ANAs), frequently occur in ADA-SCID patients after treatment. 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, new emigrant/transitional and mature naive B cells from ADA-SCID patients contained more autoreactive and ANA-expressing clones, indicative of defective central and peripheral B cell tolerance checkpoints. We further observed impaired B cell receptor (BCR) and TLR functions in B cells after ADA inhibition, which may underlie the defects in B cell tolerance. Strikingly, after HSC-GT, ADA-SCID patients displayed quasi-normal early B cell tolerance checkpoints, as evidenced by restored removal of developing autoreactive and ANA-expressing B cells. Hence, ADA plays an essential role in controlling autoreactive B cell counterselection by regulating BCR and TLR functions.
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http://dx.doi.org/10.1172/JCI61788DOI Listing
June 2012

HIV-1 envelope-dependent restriction of CXCR4-using viruses in child but not adult untransformed CD4+ T-lymphocyte lines.

Blood 2012 Mar 10;119(9):2013-23. Epub 2012 Jan 10.

AIDS Immunopathogenesis, San Raffaele Scientific Institute, Via Olgettina No. 58, Milan, Italy.

Phytohemagglutin-stimulated child and adult leukocytes equally supported CCR5-dependent (R5) and CXCR4-dependent (X4) HIV-1 replication. In contrast, when phytohemagglutin-stimulated leukocytes from either healthy or congenitally immunodeficient children were cultured on feeder cells, they well supported R5, but not X4 HIV-1 replication, whereas both viruses equally spread in adult cells maintained in similar conditions. Both child and adult cells showed similar levels of proliferation and surface expression of CD4, CCR5, CXCR4, CD25, CD69, and HLA-DR. Lack of X4 HIV-1 replication in child versus adult cells was not caused by a differential expression of several known HIV-1 restriction factors. Similar levels of HIV DNA synthesis occurred in child cells infected with R5 and X4 viruses up to 48 hours after infection when R5 HIV-1 showed a significantly superior capacity to spread in culture than X4 virus. Cultured child cells well supported single round vescicular stomatitis virus-G pseudotyped virus replication, whereas superinfection of R5-infected cells with X4 HIV-1 (or vice versa) rescued the replication of this latter virus. Thus, child cells exposed to feeder cell culture represent a novel model system in which the superior capacity of R5 versus X4 viruses to spread can be investigated in primary, untransformed CD4(+) cells.
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http://dx.doi.org/10.1182/blood-2010-12-325308DOI Listing
March 2012

Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID.

Blood 2012 Feb 19;119(6):1428-39. Epub 2011 Dec 19.

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Via Olgettina 58, Milan, Italy

Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. 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 alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.
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http://dx.doi.org/10.1182/blood-2011-07-366781DOI Listing
February 2012

In vivo T-cell dynamics during immune reconstitution after hematopoietic stem cell gene therapy in adenosine deaminase severe combined immune deficiency.

J Allergy Clin Immunol 2011 Jun 7;127(6):1368-75.e8. Epub 2011 Apr 7.

San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.

Background: Gene therapy (GT) with hematopoietic stem cells is a promising treatment for inherited immunodeficiencies.

Objectives: Limited information is available on the relative contribution of de novo thymopoiesis and peripheral expansion to T-cell reconstitution after GT as well as on the potential effects of gene transfer on hematopoietic stem cells and lymphocyte replicative lifespan. We studied these issues in patients affected by adenosine deaminase severe combined immune deficiency after low-intensity conditioning and reinfusion of retrovirally transduced autologous CD34(+) cells.

Methods: Immunophenotype, proliferative status, telomere length, and T-cell receptor excision circles were investigated at early and late time points (up to 9 years) after GT treatment. Control groups consisted of pediatric healthy donors and patients undergoing allogeneic bone marrow transplantation (BMT).

Results: We observed no telomere shortening in the bone marrow compartment and in granulocytes, whereas peripheral blood naive T cells from both GT and BMT patients showed a significant reduction in telomere length compared with healthy controls. This was in agreement with the presence of a high fraction of actively cycling naive and memory T cells and lower T-cell receptor excision circles.

Conclusion: These data indicate that T-cell homeostatic expansion contributes substantially to immune reconstitution, like BMT, and is not associated with senescence in the stem cell compartment.
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http://dx.doi.org/10.1016/j.jaci.2011.03.004DOI Listing
June 2011

Integration profile of retroviral vector in gene therapy treated patients is cell-specific according to gene expression and chromatin conformation of target cell.

EMBO Mol Med 2011 Feb 17;3(2):89-101. Epub 2011 Jan 17.

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milano, Italy.

The analysis of genomic distribution of retroviral vectors is a powerful tool to monitor 'vector-on-host' effects in gene therapy (GT) trials but also provides crucial information about 'host-on-vector' influences based on the target cell genetic and epigenetic state. We had the unique occasion to compare the insertional profile of the same therapeutic moloney murine leukemia virus (MLV) vector in the context of the adenosine deaminase-severe combined immunodeficiency (ADA-SCID) genetic background in two GT trials based on infusions of transduced mature lymphocytes (peripheral blood lymphocytes, PBL) or a single infusion of haematopoietic stem/progenitor cells (HSC). We found that vector insertions are cell-specific according to the differential expression profile of target cells, favouring, in PBL-GT, genes involved in immune system and T-cell functions/pathways as well as T-cell DNase hypersensitive sites, differently from HSC-GT. Chromatin conformations and histone modifications influenced integration preferences but we discovered that only H3K27me3 was cell-specifically disfavoured, thus representing a key epigenetic determinant of cell-type dependent insertion distribution. Our study shows that MLV vector insertional profile is cell-specific according to the genetic/chromatin state of the target cell both in vitro and in vivo in patients several years after GT.
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http://dx.doi.org/10.1002/emmm.201000108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3060339PMC
February 2011

Update on gene therapy for adenosine deaminase-deficient severe combined immunodeficiency.

Curr Opin Allergy Clin Immunol 2010 Dec;10(6):551-6

San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.

Purpose Of Review: The present review describes the recent progress in the treatment of adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) using autologous gene-modified hematopoietic stem cells, comparing immune reconstitution with respect to allogeneic transplant and discussing differences with gene therapy for SCID-X1.

Recent Findings: Since 2000, more than 30 ADA-SCID patients have been treated with gene therapy worldwide, with successful outcome in most cases, consisting of progressive immune reconstitution, efficient systemic detoxification, and long-term multilineage engraftment. Gene therapy resulted in restoration of thymic activity and T-cell functions, although the kinetic of reconstitution was slower compared with standard bone marrow transplant. Unlike allogeneic transplant from alternative donors, survival after gene therapy is excellent. In comparison with SCID-X1, ADA-SCID gene therapy presents a better safety profile and engraftment of multilineage transduced stem/progenitor cells, thanks to the use of nonmyeloablative preconditioning. New approaches using safer integrating vectors are being developed, which may lead to safer and effective gene therapy for ADA-SCID and other genetic disorders.

Summary: In the last decade, gene therapy has been developed as a successful and safe alternative strategy for patients affected by ADA-SCID lacking a compatible sibling donor. The application of innovative vector technology might further improve its efficacy and safety profile.
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http://dx.doi.org/10.1097/ACI.0b013e32833fea85DOI Listing
December 2010

Role of reduced intensity conditioning in T-cell and B-cell immune reconstitution after HLA-identical bone marrow transplantation in ADA-SCID.

Haematologica 2010 Oct 11;95(10):1778-82. Epub 2010 May 11.

HSR-TIGET, Istituto Scientifico HS Raffaele, via Olgettina 58, Milan, Italy.

The treatment of choice for severe combined immunodeficiency is bone marrow transplantation from an HLA-identical donor sibling without conditioning. However, this may result in low donor stem cell chimerism, leading to reduced long-term immune reconstitution. We compared engraftment, metabolic, and T-cell and B-cell immune reconstitution of HLA-identical sibling bone marrow transplantation performed in 2 severe combined immunodeficiency infants with adenosine deaminase deficiency from the same family treated with or without a reduced intensity conditioning regimen (busulfan/fludarabine). Only the patient who received conditioning showed a stable mixed chimerism in all lineages, including bone marrow myeloid and B cells. The use of conditioning resulted in higher thymus-derived naïve T cells and T-cell receptor excision circles, normalization of the T-cell repertoire, and faster and complete B-cell and metabolic reconstitution. These results suggest the utility of exploring the use of reduced intensity conditioning in bone marrow transplantation from HLA-identical donor in severe combined immunodeficiency to improve long-term immune reconstitution.
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http://dx.doi.org/10.3324/haematol.2010.025098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2948105PMC
October 2010

Hematopoietic stem cell gene therapy for adenosine deaminase deficient-SCID.

Immunol Res 2009 ;44(1-3):150-9

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) and Pediatric Immunohematology and Bone Marrow Transplant Unit, Scientific Institute HS Raffaele, Via Olgettina 58, 20132, Milan, Italy.

Gene therapy is a highly attractive strategy for many types of inherited disorders of the immune system. Adenosine deaminase (ADA) deficient-severe combined immunodeficiency (SCID) has been the target of several clinical trials based on the use of hematopoietic stem/progenitor cells engineered with retroviral vectors. The introduction of a low intensity conditioning regimen has been a crucial factor in achieving stable engrafment of hematopoietic stem cells and therapeutic levels of ADA-expressing cells. Recent studies have demonstrated that gene therapy for ADA-SCID has favorable safety profile and is effective in restoring normal purine metabolism and immune functions. Stem cell gene therapy combined with appropriate conditioning regimens might be extended to other genetic disorders of the hematopoietic system.
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http://dx.doi.org/10.1007/s12026-009-8107-8DOI Listing
September 2009
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