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1.
J Exp Med ; 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601675

RESUMO

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 CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 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.

2.
Blood Adv ; 3(12): 1916-1924, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31239246

RESUMO

Unveiling the mechanisms and the cellular dynamics at the basis of human hematopoietic homeostasis has been a main focus for the scientific community since the discovery of a pool of multipotent hematopoietic stem cells (HSCs) capable of sustaining the hematopoietic output throughout life and after transplantation. Recently, new works shed light on the (1) differentiation paths, (2) size and replication rate of human HSC population at steady state, and (3) role of the distinct subpopulations comprising the hematopoietic stem and progenitor cell reservoir after transplantation. These papers exploited cutting-edge technologies, including vector integration site clonal tracking, spontaneous mutations, and deep transcriptome profiling. Here we discuss the latest updates in human hematopoietic system biology and in vivo dynamics, highlighting novel concepts and common findings deriving from different approaches and the future directions of these studies. Taken together, this information contributed to partially resolving the complexity of the in vivo HSC behavior and has major implications for HSC transplantation and gene therapy as well as for the development of future therapies.

3.
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
4.
Nat Med ; 24(11): 1683-1690, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30275570

RESUMO

Hematopoietic stem and progenitor cells (HSPC) are endowed with the role of generating and maintaining lifelong the extremely diverse pool of blood cells1. Clinically, transplantation of human HSPC from an allogeneic healthy donor or infusion of autologous gene-corrected HSPC can effectively replenish defective blood cell production caused by congenital or acquired disorders2-9. However, due to methodological and ethical constraints that have limited the study of human HSPC primarily to in vitro assays10 or xenotransplantation models11,12, the in vivo activity of HSPC has to date remained relatively unexplored in humans13-16. Here we report a comprehensive study of the frequencies, dynamics and output of seven HSPC subtypes in humans that was performed by tracking 148,093 individual clones in six patients treated with lentiviral gene therapy using autologous HSPC transplantation and followed for up to 5 years. We discovered that primitive multipotent progenitor and hematopoietic stem cell (HSC) populations have distinct roles during the initial reconstitution after transplant, compared with subsequent steady-state phases. Furthermore, we showed that a fraction of in vitro-activated HSC are resilient and undergo a defined delayed activation period upon transplant. Finally, our data support the concept that early lymphoid-biased progenitors might be capable of long-term survival, such that they can be maintained independently of their continuous production from HSC. Overall, this study provides comprehensive data on HSPC dynamics after autologous transplantation and gene therapy in humans.

5.
Blood ; 132(22): 2362-2374, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30254128

RESUMO

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 ARPC1B 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 ARPC1B 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 ARPC1B 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.

6.
Cytometry A ; 91(10): 952-965, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28609016

RESUMO

Human hematopoiesis is a complex and dynamic system where morphologically and functionally diverse mature cell types are generated and maintained throughout life by bone marrow (BM) Hematopoietic Stem/Progenitor Cells (HSPC). Congenital and acquired hematopoietic disorders are often diagnosed through the detection of aberrant frequency or composition of hematopoietic cell populations. We here describe a novel protocol, called "Whole Blood Dissection" (WBD), capable of analyzing in a single test-tube, hematopoietic progenitors and all major mature cell lineages composing either BM or peripheral blood (PB) through a multiparametric flow-cytometry analysis. WBD allows unambiguously identifying in the same tube up to 23 different blood cell types including HSPC subtypes and all the major myeloid and lymphoid lineage compartments at different stages of maturation, through a combination of 17 surface and 1 viability cell markers. We assessed the efficacy of WBD by analyzing BM and PB samples from adult (n = 8) and pediatric (n = 9) healthy donors highlighting age-related shift in cell composition. We also tested the capability of WBD on detecting aberrant hematopoietic cell composition in clinical samples of patients with primary immunodeficiency or leukemia unveiling expected and novel hematopoietic unbalances. Overall, WBD allows unambiguously identifying >99% of the cell subpopulations composing a blood sample in a reproducible, standardized, cost-, and time-efficient manner. This tool has a wide range of potential pre-clinical and clinical applications going from the characterization of hematopoietic disorders to the monitoring of hematopoietic reconstitution in patients after transplant or gene therapy. © 2017 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.


Assuntos
Células Sanguíneas/citologia , Células-Tronco Hematopoéticas/citologia , Adulto , Biomarcadores/metabolismo , Células Sanguíneas/metabolismo , Linhagem da Célula/fisiologia , Criança , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Síndromes de Imunodeficiência/metabolismo , Síndromes de Imunodeficiência/patologia , Leucemia/metabolismo , Leucemia/patologia
7.
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
8.
Curr Gene Ther ; 16(3): 184-93, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27121108

RESUMO

Over the past years, clonal tracking has gained the center stage as a unique technology capable to unveil population dynamics and hierarchical relationships in vivo. We here highlighted the main open questions related to the in vivo clonal behavior of hematopoietic cells with a particular focus on hematopoietic stem and progenitor cells and T cells as main targets of cell- and gene-therapies. We walked through the current methods applied for tracing in vivo dynamics and functions of hematopoietic cells in animal models and we described the results of early studies conducted on humans. We specifically focused our attention on the recent use of retroviral/lentiviral vector Integration Site (IS) analyses to follow stably marked clones and their progeny in vivo. We showed how this molecular tracking method can be successfully employed in human studies to unveil the clonal behavior of hematopoietic cells, describing pioneering works conducted on samples from gene therapy treated patients. Clonal tracking through IS identification still comes with a complex wet-experimental protocol and technical/analytical constraints. In this regard, we reviewed the features of the available computational tools for the identification and quantification of ISs and we highlighted the potential future improvements of IS-based tracking, as this technology is becoming a major source of information on in vivo fate and survival of engineered cells in humans.


Assuntos
Células-Tronco Hematopoéticas , Ensaios de Triagem em Larga Escala/métodos , Animais , Células Clonais , Biologia Computacional/métodos , Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Subpopulações de Linfócitos T/fisiologia
9.
Sci Transl Med ; 7(273): 273ra13, 2015 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-25653219

RESUMO

A definitive understanding of survival and differentiation potential in humans of T cell subpopulations is of paramount importance for the development of effective T cell therapies. In particular, uncovering the dynamics in vivo in humans of the recently described T memory stem cells (TSCM) would be crucial for therapeutic approaches that aim at taking advantage of a stable cellular vehicle with precursor potential. We exploited data derived from two gene therapy clinical trials for an inherited immunodeficiency, using either retrovirally engineered hematopoietic stem cells or mature lymphocytes to trace individual T cell clones directly in vivo in humans. We compared healthy donors and bone marrow-transplanted patients, studied long-term in vivo T cell composition under different clinical conditions, and specifically examined TSCM contribution according to age, conditioning regimen, disease background, cell source, long-term reconstitution, and ex vivo gene correction processing. High-throughput sequencing of retroviral vector integration sites (ISs) allowed tracing the fate of more than 1700 individual T cell clones in gene therapy patients after infusion of gene-corrected hematopoietic stem cells or mature lymphocytes. We shed light on long-term in vivo clonal relationships among different T cell subtypes, and we unveiled that TSCM are able to persist and to preserve their precursor potential in humans for up to 12 years after infusion of gene-corrected lymphocytes. Overall, this work provides high-resolution tracking of T cell fate and activity and validates, in humans, the safe and functional decade-long survival of engineered TSCM, paving the way for their future application in clinical settings.


Assuntos
Rastreamento de Células , Engenharia Genética , Células-Tronco Hematopoéticas/citologia , Memória Imunológica , Linfócitos T/citologia , Linfócitos T/metabolismo , Adulto , Sobrevivência Celular , Criança , Células Clonais , Terapia Genética , Humanos , Interleucina-2/farmacologia , Estudos Longitudinais , Subpopulações de Linfócitos/metabolismo , Fenótipo , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Doadores de Tecidos
10.
PLoS One ; 6(9): e25162, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21966443

RESUMO

BACKGROUND: The differentiation program of thyroid follicular cells (TFCs), by far the most abundant cell population of the thyroid gland, relies on the interplay between sequence-specific transcription factors and transcriptional coregulators with the basal transcriptional machinery of the cell. However, the molecular mechanisms leading to the fully differentiated thyrocyte are still the object of intense study. The transcription factor Pax8, a member of the Paired-box gene family, has been demonstrated to be a critical regulator required for proper development and differentiation of thyroid follicular cells. Despite being Pax8 well-characterized with respect to its role in regulating genes involved in thyroid differentiation, genomics approaches aiming at the identification of additional Pax8 targets are lacking and the biological pathways controlled by this transcription factor are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: To identify unique downstream targets of Pax8, we investigated the genome-wide effect of Pax8 silencing comparing the transcriptome of silenced versus normal differentiated FRTL-5 thyroid cells. In total, 2815 genes were found modulated 72 h after Pax8 RNAi, induced or repressed. Genes previously reported to be regulated by Pax8 in FRTL-5 cells were confirmed. In addition, novel targets genes involved in functional processes such as DNA replication, anion transport, kinase activity, apoptosis and cellular processes were newly identified. Transcriptome analysis highlighted that Pax8 is a key molecule for thyroid morphogenesis and differentiation. CONCLUSIONS/SIGNIFICANCE: This is the first large-scale study aimed at the identification of new genes regulated by Pax8, a master regulator of thyroid development and differentiation. The biological pathways and target genes controlled by Pax8 will have considerable importance to understand thyroid disease progression as well as to set up novel therapeutic strategies.


Assuntos
Inativação Gênica/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fatores de Transcrição Box Pareados/metabolismo , Glândula Tireoide/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Imunoprecipitação da Cromatina , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados/genética , Interferência de RNA , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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