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Vaccination with Bacillus Calmette–Guérin (BCG) can be harmful to patients with combined primary immunodeficiencies. We report the outcome of BCG vaccination in a series of twelve patients affected by adenosine deaminase deficiency (ADA-SCID). BCG vaccination resulted in a very high incidence of complications due to uncontrolled replication of the mycobacterium. All patients who developed BCG-related disease were treated successfully and remained free from recurrence of disease. We recommend the prompt initiation of enzyme replacement therapy and secondary prophylaxis to reduce the risk of BCG-related complications in ADA-SCID patients.

Collection of an adequate amount of autologous haematopoietic stem progenitor cells (HSPC) is required for ex vivo manipulation and successful engraftment for certain inherited disorders. Fifty-seven paediatric patients (age 0.5–11.4 years) underwent a bone marrow harvest for the purpose of HSPC gene therapy (GT), including adenosine deaminase-severe combined immunodeficiency (ADA-SCID), Wiskott–Aldrich syndrome (WAS) and metachromatic leukodystrophy (MLD) patients. Total nucleated cells and the percentage and absolute counts of CD34+ cells were calculated at defined steps of the procedure (harvest, CD34+ cell purification, transduction with the gene transfer vector and infusion of the medicinal product). A minimum CD34+ cell dose for infusion was 2 × 106/kg, with an optimal target at 5–10 × 106/kg. Median volume of bone marrow harvested was 34.2 ml/kg (range 14.2–56.6). The number of CD34+ cells collected correlated inversely with weight and age in all patients and particularly in the MLD children group. All patients reached the minimum target dose for infusion: median dose of CD34+ cells/kg infused was 10.3 × 106/kg (3.7–25.9), with no difference among the three groups. Bone marrow harvest of volumes > 30 ml/kg in infants and children with ADA-SCID, WAS and MLD is well tolerated and allows obtaining an adequate dose of a medicinal product for HSPC-GT.

Patients with inborn error diseases can be candidates for autologous haematopoietic stem cells (HSC) gene therapies (GT) but may require negative viral screening, including Hepatitis C (HCV), to allow HSC manipulation in Good Manufacturing Practices areas. In case of HCV positivity, patients might be excluded from life-saving treatments. As HCV antibodies could be negative in young infant immunodeficient patients due to their immature/impaired immune system, or positive due to maternal-fetal antibody transmission, the risk is usually also evaluated on the basis of the HCV-RNA. This article is protected by copyright. All rights reserved.

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.

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.

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

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.

Next-Generation Gene Therapy Few disciplines in contemporary clinical research have experienced the high expectations directed at the gene therapy field. However, gene therapy has been challenging to translate to the clinic, often because the therapeutic gene is expressed at insufficient levels in the patient or because the gene delivery vector integrates near protooncogenes, which can cause leukemia (see the Perspective by Verma ). Biffi et al. ( 1233158 , published online 11 July) and Aiuti et al. ( 1233151 ; published online 11 July) report progress on both fronts in gene therapy trials of three patients with metachromatic leukodystrophy (MLD), a neurodegenerative disorder, and three patients with Wiskott-Aldrich syndrome (WAS), an immunodeficiency disorder. Optimized lentiviral vectors were used to introduce functional MLD or WAS genes into the patients' hematopoietic stem cells (HSCs) ex vivo, and the transduced cells were then infused back into the patients, who were then monitored for up to 2 years. In both trials, the patients showed stable engraftment of the transduced HSC and high expression levels of functional MLD or WAS genes. Encouragingly, there was no evidence of lentiviral vector integration near proto-oncogenes, and the gene therapy treatment halted disease progression in most patients. A longer follow-up period will be needed to further validate efficacy and safety.

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients. Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients. "\"\\\"\\\\\\\"\\\\\\\\\\\\\\\"\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\"\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\"Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients.\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\"\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\"\\\\\\\\\\\\\\\"\\\\\\\"\\\"\""

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. (J Allergy Clin Immunol 2011;127:1368-75.) 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.

Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report 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-κB 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. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, recurrent infections, eczema, autoimmunity and increased susceptibility to malignancies. Allogeneic hematopoietic stem cell transplantation (HSCT) is a recognized curative treatment for WAS, but is still associated with transplant-related complications and long-term morbidity, particularly in the absence of fully matched donors. In April 2010, we initiated a phase I/II clinical trial with hematopoietic stem cell (HSC) gene therapy (GT) for WAS. The investigational medicinal product (IMP) consists of autologous CD34+ HSC engineered with a lentiviral vector (LV) driving the expression of WAS cDNA from an endogenous 1.6 kb human WAS promoter (LV-WAS), infused after a reduced intensity conditioning (RIC) based on anti-CD20 mAb, targeted busulfan and fludarabine. We previously reported early follow up (FU) results from the first 3 patients (Aiuti et al., Science 2013). Seven patients (Zhu score ≥3) have now been treated at a median age of 1.9 years (1.1 - 11.1). As of May 2015, all patients are alive with a median FU of 3.2 years (0.7 - 5.0). CD34+ cell source was bone marrow (BM) (n=5), mobilized peripheral blood (MPB) (n=1) or both (n=1). IMP dose ranged between 7.0 and 14.1 x106 CD34+/kg, containing on average 94.4 ± 3.5% transduced clonogenic progenitors and a mean vector copy number (VCN)/genome in bulk CD34+ cells of 2.7 ± 0.8. No adverse reactions were observed after IMP infusion and RIC was well tolerated. Median duration of severe neutropenia was 19 days; granulocyte-colony stimulating factor was administered to 1 patient. In the first 6 treated patients with FU >2 years, we observed robust and persistent engraftment of gene corrected cells. At the most recent FU, transduced BM progenitors ranged between 20.7 and 59.7%, and LV-transduced cells were detected in multiple lineages, including PB granulocytes (VCN 0.34 - 0.93) and lymphocytes (VCN 1.18 - 2.73). WAS protein expression, measured by flow-cytometry, was detected in the majority of PB platelets [mean ± standard deviation (SD), 71.4 ± 14.0%], monocytes (63.3 ± 18.5%) and lymphocytes (78.9 ± 14.9%). Lymphocyte subset counts were normal in most patients and proliferative response to anti-CD3 mAb was in the normal range in all 6 patients. After immune reconstitution, a marked reduction in the annualized estimated rate of severe infections was observed, as compared with baseline (figure 1A). The first 6 treated patients discontinued anti-infective prophylaxis and no longer require a protected environment. Four patients stopped immunoglobulin supplementation and 2 of them developed specific antibodies after vaccination. Eczema resolved in 4 patients and remains mild in 2. No clinical manifestations of autoimmunity were observed ≥1 year after GT in accordance with improved B-cell development and decreased autoantibody production. All patients became platelet transfusion independent at a median of 4 months after GT (range: 1.0 - 8.7). Mean platelet counts progressively increased after treatment (mean ± SD: before GT, 13.4 ± 7.8 x109/l; 24-30 month FU, 45.8 ± 22.0 x109/l; 36-42 month FU, 57.0 ± 18.7 x109/l). The frequency and the severity of bleeding events decreased after the 1st year of FU. No severe bleedings were recorded after treatment (figure 1B). Quality of life improved in all patients after GT. From the 2nd year of FU, the number of hospitalizations for infections decreased and no hospitalizations due to bleeding were observed after treatment. The seventh patient treated, who received MPB derived CD34+ cells only, showed the fastest platelet recovery with the highest level of transduced myeloid cell engraftment, and is clinically well. No Serious Adverse Events (SAE) related to the IMP were observed. The most frequent SAE were related to infections (85%), occuring mainly during the 1st year of FU. Importantly, no evidence of abnormal clonal proliferations emerged after GT and the LV integration profile show a polyclonal pattern, with no skewing for proto-oncogenes. In conclusion, this updated report in 7 WAS patients show that GT is well tolerated and leads to a sustained clinical benefit. The high level of gene transfer obtained with LV-WAS results in robust engraftment of transduced HSC, even when combined with RIC. Prolonged FU will provide additional information on the long-term safety and clinical efficacy of this treatment. Figure 1. Figure 1. Disclosures Villa: Fondazione Telethon: Research Funding. Dott:GlaxoSmithKline: Consultancy. van Rossem:GlaxoSmithKline: Employment. Naldini:Salk Institute: Patents & Royalties: Lentiviral vectors; San Raffaele Telethon Institute: Patents & Royalties: Lentiviral vector technology; GlaxoSmithKline: Other: GSK licensed gene therapies developed at my Institute and the Institute receives milestone payments; Sangamo Biosciences: Research Funding; Biogen: Research Funding; Genenta Sciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Aiuti:GlaxoSmithKline (GSK): Other: PI of clinical trial which is financially sponsored by GSK; Fondazione Telethon: Research Funding.

Adenosine deaminase (ADA)-SCID is characterized by increased purine metabolites and severe combined immunodeficiency. Autoimmune manifestations have been observed in milder forms or after enzyme replacement therapy (ERT), and recently in patients following gene therapy (GT). nTregs have a full enzymatic machinery to generate and sustain high concentrations of extracellular adenosine and may be involved in the pathogenesis of autoimmunity. We analyzed the frequency of CD4+CD25+FOXP3+CD127- in the peripheral blood, the methylation profile of the FoxP3 gene and the suppressive function of nTreg in ADA-SCID patients following ERT, GT or bone marrow transplant (BMT) as compared to controls. We found that the proportion and function of nTregs after GT mirrored that of controls, while nTreg from the ERT group showed a reduced frequency and impaired function. We also investigated the maturation state of B cells in the peripheral blood of ADA-SCID patients. After ERT and early after GT, patients displayed an increased proportion of transitional B cells (CD24highCD38high) and of cells with an incomplete BCR (CD21-CD35-) as compared to BMT and healthy controls. At later follow up patients treated with GT showed a tendency to normalize the phenotype in parallel to an increased frequency of gene corrected cells. Collectively, these data provide a first indication of predisposition to autoimmunity in ADA-SCID and the role of different treatments in the maintenance of peripheral tolerance.