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1.
Lancet Haematol ; 6(5): e239-e253, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30981783

RESUMO

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 × 109 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 × 109 per L in one patient, 50-100 × 109 per L in five patients, and more than 100 × 109 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.


Assuntos
Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Criança , Pré-Escolar , Feminino , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Lactente , Itália , Masculino , Mutação , Linfócitos T/imunologia , Linfócitos T/metabolismo , Condicionamento Pré-Transplante/métodos , Resultado do Tratamento , Síndrome de Wiskott-Aldrich/sangue , Síndrome de Wiskott-Aldrich/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/genética
2.
J Clin Invest ; 129(4): 1566-1580, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30830876

RESUMO

BACKGROUND: The human bone marrow (BM) niche contains a population of mesenchymal stromal cells (MSCs) that provide physical support and regulate hematopoietic stem cell (HSC) homeostasis. ß-Thalassemia (BT) is a hereditary disorder characterized by altered hemoglobin beta-chain synthesis amenable to allogeneic HSC transplantation and HSC gene therapy. Iron overload (IO) is a common complication in BT patients affecting several organs. However, data on the BM stromal compartment are scarce. METHODS: MSCs were isolated and characterized from BM aspirates of healthy donors (HDs) and BT patients. The state of IO was assessed and correlated with the presence of primitive MSCs in vitro and in vivo. Hematopoietic supportive capacity of MSCs was evaluated by transwell migration assay and 2D coculture of MSCs with human CD34+ HSCs. In vivo, the ability of MSCs to facilitate HSC engraftment was tested in a xenogenic transplant model, whereas the capacity to sustain human hematopoiesis was evaluated in humanized ossicle models. RESULTS: We report that, despite iron chelation, BT BM contains high levels of iron and ferritin, indicative of iron accumulation in the BM niche. We found a pauperization of the most primitive MSC pool caused by increased ROS production in vitro which impaired MSC stemness properties. We confirmed a reduced frequency of primitive MSCs in vivo in BT patients. We also discovered a weakened antioxidative response and diminished expression of BM niche-associated genes in BT-MSCs. This caused a functional impairment in MSC hematopoietic supportive capacity in vitro and in cotransplantation models. In addition, BT-MSCs failed to form a proper BM niche in humanized ossicle models. CONCLUSION: Our results suggest an impairment in the mesenchymal compartment of BT BM niche and highlight the need for novel strategies to target the niche to reduce IO and oxidative stress before transplantation. FUNDING: This work was supported by the SR-TIGET Core grant from Fondazione Telethon and by Ricerca Corrente.

3.
Nat Med ; 25(2): 234-241, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30664781

RESUMO

ß-thalassemia is caused by ß-globin gene mutations resulting in reduced (ß+) or absent (ß0) hemoglobin production. Patient life expectancy has recently increased, but the need for chronic transfusions in transfusion-dependent thalassemia (TDT) and iron chelation impairs quality of life1. Allogeneic hematopoietic stem cell (HSC) transplantation represents the curative treatment, with thalassemia-free survival exceeding 80%. However, it is available to a minority of patients and is associated with morbidity, rejection and graft-versus-host disease2. Gene therapy with autologous HSCs modified to express ß-globin represents a potential therapeutic option. We treated three adults and six children with ß0 or severe ß+ mutations in a phase 1/2 trial ( NCT02453477 ) with an intrabone administration of HSCs transduced with the lentiviral vector GLOBE. Rapid hematopoietic recovery with polyclonal multilineage engraftment of vector-marked cells was achieved, with a median of 37.5% (range 12.6-76.4%) in hematopoietic progenitors and a vector copy number per cell (VCN) of 0.58 (range 0.10-1.97) in erythroid precursors at 1 year, in absence of clonal dominance. Transfusion requirement was reduced in the adults. Three out of four evaluable pediatric participants discontinued transfusions after gene therapy and were transfusion independent at the last follow-up. Younger age and persistence of higher VCN in the repopulating hematopoietic cells are associated with better outcome.


Assuntos
Transfusão de Sangue , Osso e Ossos/patologia , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Talassemia beta/genética , Talassemia beta/terapia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Resultado do Tratamento
4.
Cell Stem Cell ; 19(1): 107-19, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27237736

RESUMO

Hematopoietic stem/progenitor cells (HSPCs) are capable of supporting the lifelong production of blood cells exerting a wide spectrum of functions. Lentiviral vector HSPC gene therapy generates a human hematopoietic system stably marked at the clonal level by vector integration sites (ISs). Using IS analysis, we longitudinally tracked >89,000 clones from 15 distinct bone marrow and peripheral blood lineages purified up to 4 years after transplant in four Wiskott-Aldrich syndrome patients treated with HSPC gene therapy. We measured at the clonal level repopulating waves, populations' sizes and dynamics, activity of distinct HSPC subtypes, contribution of various progenitor classes during the early and late post-transplant phases, and hierarchical relationships among lineages. We discovered that in-vitro-manipulated HSPCs retain the ability to return to latency after transplant and can be physiologically reactivated, sustaining a stable hematopoietic output. This study constitutes in vivo comprehensive tracking in humans of hematopoietic clonal dynamics during the early and late post-transplant phases.


Assuntos
Rastreamento de Células , Hematopoese , Antígenos CD34/metabolismo , Engenharia Celular , Linhagem da Célula/genética , Pré-Escolar , Células Clonais , Terapia Genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lactente , Masculino , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Mutagênese Insercional/genética , Fatores de Tempo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia
6.
Stem Cell Reports ; 5(4): 558-68, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26344905

RESUMO

Autosomal recessive osteopetrosis is a human bone disease mainly caused by TCIRG1 gene mutations that prevent osteoclasts resorbing activity, recapitulated by the oc/oc mouse model. Bone marrow transplantation is the only available treatment, limited by the need for a matched donor. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of autologous cells to generate gene corrected osteoclasts might represent a powerful alternative. We generated iPSCs from oc/oc mice, corrected the mutation using a BAC carrying the entire Tcirg1 gene locus as a template for homologous recombination, and induced hematopoietic differentiation. Similarly to physiologic fetal hematopoiesis, iPSC-derived CD41(+) cells gradually gave rise to CD45(+) cells, which comprised both mature myeloid cells and high proliferative potential colony-forming cells. Finally, we differentiated the gene corrected iPSC-derived myeloid cells into osteoclasts with rescued bone resorbing activity. These results are promising for a future translation into the human clinical setting.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Osteoclastos/citologia , Osteopetrose/terapia , Reparo Gênico Alvo-Dirigido/métodos , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Diferenciação Celular , Linhagem Celular , Hematopoese , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Células Mieloides/citologia , Células Mieloides/metabolismo , Osteoclastos/metabolismo , Osteopetrose/genética
7.
J Clin Invest ; 125(10): 3941-51, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26368308

RESUMO

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by microthrombocytopenia, eczema, and high susceptibility to developing tumors and autoimmunity. Recent evidence suggests that B cells may be key players in the pathogenesis of autoimmunity in WAS. Here, we assessed whether WAS protein deficiency (WASp deficiency) affects the establishment of B cell tolerance by testing the reactivity of recombinant antibodies isolated from single B cells from 4 WAS patients before and after gene therapy (GT). We found that pre-GT WASp-deficient B cells were hyperreactive to B cell receptor stimulation (BCR stimulation). This hyperreactivity correlated with decreased frequency of autoreactive new emigrant/transitional B cells exiting the BM, indicating that the BCR signaling threshold plays a major role in the regulation of central B cell tolerance. In contrast, mature naive B cells from WAS patients were enriched in self-reactive clones, revealing that peripheral B cell tolerance checkpoint dysfunction is associated with impaired suppressive function of WAS regulatory T cells. The introduction of functional WASp by GT corrected the alterations of both central and peripheral B cell tolerance checkpoints. We conclude that WASp plays an important role in the establishment and maintenance of B cell tolerance in humans and that restoration of WASp by GT is able to restore B cell tolerance in WAS patients.


Assuntos
Linfócitos B/imunologia , Terapia Genética , Vetores Genéticos/uso terapêutico , Tolerância Imunológica , Proteína da Síndrome de Wiskott-Aldrich/uso terapêutico , Síndrome de Wiskott-Aldrich/terapia , Adulto , Sequência de Aminoácidos , Medula Óssea/patologia , Criança , Pré-Escolar , Deleção Clonal , Células Clonais/imunologia , Humanos , Lentivirus/genética , Masculino , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos B/imunologia , Proteínas Recombinantes de Fusão , Linfócitos T Reguladores/imunologia , Síndrome de Wiskott-Aldrich/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética
8.
J Allergy Clin Immunol ; 136(3): 692-702.e2, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25792466

RESUMO

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.


Assuntos
Subpopulações de Linfócitos B/metabolismo , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Autoanticorpos/biossíntese , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Medula Óssea/imunologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Criança , Pré-Escolar , Expressão Gênica , Perfilação da Expressão Gênica , Vetores Genéticos , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoglobulinas/biossíntese , Imunofenotipagem , Lactente , Lentivirus/genética , Masculino , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução Genética , Condicionamento Pré-Transplante , Transplante Autólogo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia
9.
Mol Ther ; 22(8): 1472-1483, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24869932

RESUMO

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.


Assuntos
Terapia Genética/métodos , Doença Granulomatosa Crônica/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/virologia , Glicoproteínas de Membrana/metabolismo , MicroRNAs/genética , NADPH Oxidases/metabolismo , Animais , Antígenos CD34/metabolismo , Linhagem Celular , Células Cultivadas , Terapia Combinada , Modelos Animais de Doenças , Vetores Genéticos/uso terapêutico , Doença Granulomatosa Crônica/patologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lentivirus/genética , Camundongos , Células Mieloides/metabolismo , NADPH Oxidase 2
10.
J Allergy Clin Immunol ; 134(2): 420-8, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24679343

RESUMO

BACKGROUND: Five different G protein-coupled sphingosine-1-phosphate (S1P) receptors (S1P1-S1P5) regulate a variety of physiologic and pathophysiologic processes, including lymphocyte circulation, multiple sclerosis (MS), and cancer. Although B-lymphocyte circulation plays an important role in these processes and is essential for normal immune responses, little is known about S1P receptors in human B cells. OBJECTIVE: To explore their function and signaling, we studied B-cell lines and primary B cells from control subjects, patients with leukemia, patients with S1P receptor inhibitor-treated MS, and patients with primary immunodeficiencies. METHODS: S1P receptor expression was analyzed by using multicolor immunofluorescence microscopy and quantitative PCR. Transwell assays were used to study cell migration. S1P receptor internalization was visualized by means of time-lapse imaging with fluorescent S1P receptor fusion proteins expressed by using lentiviral gene transfer. B-lymphocyte subsets were characterized by means of flow cytometry and immunofluorescence microscopy. RESULTS: Showing that different B-cell populations express different combinations of S1P receptors, we found that S1P1 promotes migration, whereas S1P4 modulates and S1P2 inhibits S1P1 signals. Expression of CD69 in activated B lymphocytes and B cells from patients with chronic lymphocytic leukemia inhibited S1P-induced migration. Studying B-cell lines, normal B lymphocytes, and B cells from patients with primary immunodeficiencies, we identified Bruton tyrosine kinase, ß-arrestin 2, LPS-responsive beige-like anchor protein, dedicator of cytokinesis 8, and Wiskott-Aldrich syndrome protein as critical signaling components downstream of S1P1. CONCLUSION: Thus S1P receptor signaling regulates human B-cell circulation and might be a factor contributing to the pathology of MS, chronic lymphocytic leukemia, and primary immunodeficiencies.


Assuntos
Subpopulações de Linfócitos B/metabolismo , Imunodeficiência de Variável Comum/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Esclerose Múltipla/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Tirosina Quinase da Agamaglobulinemia , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Arrestinas/genética , Arrestinas/imunologia , Arrestinas/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Linhagem Celular , Movimento Celular , Imunodeficiência de Variável Comum/genética , Imunodeficiência de Variável Comum/imunologia , Imunodeficiência de Variável Comum/patologia , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Cultura Primária de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/imunologia , Transdução de Sinais , Imagem com Lapso de Tempo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/imunologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , beta-Arrestina 2 , beta-Arrestinas
11.
J Inherit Metab Dis ; 37(4): 525-33, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24619149

RESUMO

In the last years important progress has been made in the treatment of several primary immunodeficiency disorders (PIDs) with gene therapy. Hematopoietic stem cell (HSC) gene therapy indeed represents a valid alternative to conventional transplantation when a compatible donor is not available and recent success confirmed the great potential of this approach. First clinical trials performed with gamma retroviral vectors were promising and guaranteed clinical benefits to the patients. On the other hand, the outcome of severe adverse events as the development of hematological abnormalities highlighted the necessity to develop a safer platform to deliver the therapeutic gene. Self-inactivating (SIN) lentiviral vectors (LVVs) were studied to overcome this hurdle through their preferable integration pattern into the host genome. In this review, we describe the recent advancements achieved both in vitro and at preclinical level with LVVs for the treatment of Wiskott-Aldrich syndrome (WAS), chronic granulomatous disease (CGD), ADA deficiency (ADA-SCID), Artemis deficiency, RAG1/2 deficiency, X-linked severe combined immunodeficiency (γchain deficiency, SCIDX1), X-linked lymphoproliferative disease (XLP) and immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome.


Assuntos
Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Síndromes de Imunodeficiência/terapia , Lentivirus/genética , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Agamaglobulinemia/genética , Agamaglobulinemia/terapia , Animais , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/terapia , Humanos , Síndromes de Imunodeficiência/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia
12.
J Allergy Clin Immunol ; 133(3): 799-806.e10, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24506932

RESUMO

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.


Assuntos
Adenosina Desaminase/deficiência , Linfócitos B/fisiologia , Terapia de Reposição de Enzimas , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Adenosina Desaminase/genética , Adenosina Desaminase/uso terapêutico , Adolescente , Fator Ativador de Células B/fisiologia , Linfócitos B/imunologia , Criança , Pré-Escolar , Humanos , Lactente
13.
J Autoimmun ; 50: 42-50, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24369837

RESUMO

Wiskott-Aldrich Syndrome protein (WASp) regulates the cytoskeleton in hematopoietic cells and mutations in its gene cause the Wiskott-Aldrich Syndrome (WAS), a primary immunodeficiency with microthrombocytopenia, eczema and a higher susceptibility to develop tumors. Autoimmune manifestations, frequently observed in WAS patients, are associated with an increased risk of mortality and still represent an unsolved aspect of the disease. B cells play a crucial role both in immune competence and self-tolerance and defects in their development and function result in immunodeficiency and/or autoimmunity. We performed a phenotypical and molecular analysis of central and peripheral B-cell compartments in WAS pediatric patients. We found a decreased proportion of immature B cells in the bone marrow correlating with an increased presence of transitional B cells in the periphery. These results could be explained by the defective migratory response of WAS B cells to SDF-1α, essential for the retention of immature B cells in the BM. In the periphery, we observed an unusual expansion of CD21(low) B-cell population and increased plasma BAFF levels that may contribute to the high susceptibility to develop autoimmune manifestations in WAS patients. WAS memory B cells were characterized by a reduced in vivo proliferation, decreased somatic hypermutation and preferential usage of IGHV4-34, an immunoglobulin gene commonly found in autoreactive B cells. In conclusion, our findings demonstrate that WASp-deficiency perturbs B-cell homeostasis thus adding a new layer of immune dysregulation concurring to the increased susceptibility to develop autoimmunity in WAS patients.


Assuntos
Autoimunidade , Linfócitos B/imunologia , Suscetibilidade a Doenças/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Síndrome de Wiskott-Aldrich/imunologia , Fator Ativador de Células B/sangue , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Linfócitos B/patologia , Medula Óssea/imunologia , Medula Óssea/patologia , Diferenciação Celular , Movimento Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/imunologia , Expressão Gênica , Homeostase/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Memória Imunológica , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/imunologia , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/imunologia
14.
Science ; 341(6148): 1233151, 2013 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-23845947

RESUMO

Wiskott-Aldrich syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative, but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and reinfused the cells after a reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical scores. Vector integration analyses revealed highly polyclonal and multilineage haematopoiesis resulting from the gene-corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.


Assuntos
Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Criança , Vetores Genéticos , Humanos , Lentivirus , Masculino , Transdução Genética , Integração Viral
15.
Mol Ther ; 21(1): 175-84, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22371846

RESUMO

Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34(+) cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34(+) cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34(+) cells gene therapy for the treatment of WAS.


Assuntos
Antígenos CD34/imunologia , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Vetores Genéticos , Lentivirus/genética , Transdução Genética , Síndrome de Wiskott-Aldrich/terapia , Animais , Células da Medula Óssea/imunologia , Camundongos , Camundongos Knockout
16.
Blood ; 119(6): 1428-39, 2012 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-22184407

RESUMO

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.


Assuntos
5'-Nucleotidase/imunologia , Adenosina/imunologia , Agamaglobulinemia/imunologia , Antígenos CD/imunologia , Apirase/imunologia , Imunodeficiência Combinada Severa/imunologia , Linfócitos T Reguladores/imunologia , 5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Adenosina Desaminase/metabolismo , Adenosina Desaminase/uso terapêutico , Adolescente , Adulto , Agamaglobulinemia/genética , Agamaglobulinemia/terapia , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Autoanticorpos/imunologia , Criança , Pré-Escolar , Feminino , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Hipotireoidismo/enzimologia , Hipotireoidismo/genética , Hipotireoidismo/imunologia , Imuno-Histoquímica , Lactente , Masculino , Camundongos , Camundongos Knockout , Polietilenoglicóis/química , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Linfócitos T Reguladores/metabolismo
18.
J Allergy Clin Immunol ; 127(6): 1368-75.e8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21477850

RESUMO

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.


Assuntos
Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Subpopulações de Linfócitos T/imunologia , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Adulto , Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Agamaglobulinemia/terapia , Transplante de Medula Óssea , Estudos de Casos e Controles , Ciclo Celular , Senescência Celular/genética , Criança , Granulócitos/patologia , Humanos , Memória Imunológica , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Retroviridae/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/terapia , Subpopulações de Linfócitos T/patologia , Telômero/genética , Transplante Autólogo , Transplante Homólogo
19.
J Allergy Clin Immunol ; 125(2): 439-448.e8, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20159256

RESUMO

BACKGROUND: The Wiskott-Aldrich syndrome (WAS) is a rare genetic disease characterized by thrombocytopenia, immunodeficiency, autoimmunity, and hematologic malignancies. Secondary mutations leading to re-expression of WAS protein (WASP) are relatively frequent in patients with WAS. OBJECTIVE: The tissue distribution and function of revertant cells were investigated in a novel case of WAS gene secondary mutation. METHODS: A vast combination of approaches was used to characterize the second-site mutation, to investigate revertant cell function, and to track their distribution over a 18-year clinical follow-up. RESULTS: The WAS gene secondary mutation was a 4-nucleotide insertion, 4 nucleotides downstream of the original deletion. This somatic mutation allowed the T-cell-restricted expression of a stable, full-length WASP with a 3-amino acid change compared with the wild-type protein. WASP(+) T cells appeared early in the spleen (age 10 years) and were highly enriched in a mesenteric lymph node at a later time (age 23 years). Revertant T cells had a diversified T-cell-receptor repertoire and displayed in vitro and in vivo selective advantage. They proliferated and produced cytokines normally on T-cell-receptor stimulation. Consistently, the revertant WASP correctly localized to the immunologic synapse and to the leading edge of migrating T cells. CONCLUSION: Despite the high proportion of functional revertant T cells, the patient still has severe infections and autoimmune disorders, suggesting that re-expression of WASP in T cells is not sufficient to normalize immune functions fully in patients with WAS.


Assuntos
Tecido Linfoide/imunologia , Linfócitos T/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologia , Adulto , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Separação Celular , Análise Mutacional de DNA , Citometria de Fluxo , Humanos , Tecido Linfoide/citologia , Masculino , Microscopia Confocal , Dados de Sequência Molecular , Mosaicismo , Mutação , Reação em Cadeia da Polimerase
20.
Mol Ther ; 17(6): 1073-82, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19259069

RESUMO

Wiskott-Aldrich Syndrome (WAS) is a life-threatening X-linked disease characterized by immunodeficiency, thrombocytopenia, autoimmunity, and malignancies. Gene therapy could represent a therapeutic option for patients lacking a suitable bone marrow (BM) donor. In this study, we analyzed the long-term outcome of WAS gene therapy mediated by a clinically compatible lentiviral vector (LV) in a large cohort of was(null) mice. We demonstrated stable and full donor engraftment and Wiskott-Aldrich Syndrome protein (WASP) expression in various hematopoietic lineages, up to 12 months after gene therapy. Importantly, we observed a selective advantage for T and B lymphocytes expressing transgenic WASP. T-cell receptor (TCR)-driven T-cell activation, as well as B-cell's ability to migrate in response to CXCL13, was fully restored. Safety was evaluated throughout the long-term follow-up of primary and secondary recipients of WAS gene therapy. WAS gene therapy did not affect the lifespan of treated animals. Both hematopoietic and nonhematopoietic tumors arose, but we excluded the association with gene therapy in all cases. Demonstration of long-term efficacy and safety of WAS gene therapy mediated by a clinically applicable LV is a key step toward the implementation of a gene therapy clinical trial for WAS.


Assuntos
Terapia Genética/efeitos adversos , Terapia Genética/métodos , Síndrome de Wiskott-Aldrich/terapia , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Western Blotting , Feminino , Imunofenotipagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Reação em Cadeia da Polimerase , Linfócitos T/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
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