RESUMO
Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) ß chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal γ-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the HBG γ-globin promoters disrupt binding sites for the repressors BCL11A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the HBG promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in γ-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.
Assuntos
Anemia Falciforme , Talassemia beta , Anemia Falciforme/genética , Anemia Falciforme/terapia , Sítios de Ligação , Sistemas CRISPR-Cas , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Edição de Genes/métodos , Humanos , Fenótipo , Globinas beta/genética , Globinas beta/metabolismo , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/terapia , gama-Globinas/genética , gama-Globinas/metabolismoRESUMO
We report outcomes after hematopoietic stem cell transplant for three patients with X-MAID, including 1 patient from the originally described cohort and two brothers with positive TREC newborn screening for SCID who were found to have a T-B-NK+ SCID phenotype attributable to X-linked moesin associated immunodeficiency (X-MAID). A c.511C>T variant in moesin was identified via exome sequencing in the older of these siblings in the setting of low lymphocyte counts and poor proliferative responses consistent with SCID. He received reduced intensity conditioning due to CMV, and was transplanted with a T-depleted haploidentical (maternal) donor. His post-transplant course was complicated by hemolytic anemia, neutropenia, and sepsis. He had poor engraftment, requiring a 2nd transplant. His younger brother presented with the same clinical phenotype and was treated with umbilical cord blood transplant following myeloablative conditioning, has engrafted and is doing well. The third case also presented with severe lymphopenia in infancy, received a matched related bone marrow transplant following myeloablative conditioning, has engrafted and is doing well. These cases represent a novel manifestation of non-radiosensitive X-linked form of T-B-NK+ SCID that is able to be detected by TREC based newborn screening and effectively treated with HCT.
RESUMO
Pioneering gene therapy trials have shown that the genetic engineering of haematopoietic stem and progenitor cells can be an alternative to allogeneic transplantation in the treatment of primary immunodeficiencies. Early trials also highlighted the risk of insertional mutagenesis and oncogene transactivation associated with the first generation of gammaretroviral vectors. These events prompted the development of safer, self-inactivating lentiviral or gammaretroviral vectors. These lentiviral vectors have been successfully used to treat over 200 patients with 10 different haematological disorders (including primary immunodeficiencies, haemoglobinopathies and metabolic disorders) and for the generation of chimeric antigen receptor-T cells for cancer therapy. However, several challenges, such as effective reconstitution during inflammation, remain if gene therapy is to be extended to more complex diseases in which haematopoietic stem and progenitor cells can be altered by the disease environment. We discuss the progress made and future challenges for gene therapy and contrast gene therapy with gene-editing strategies.
Assuntos
Anemia Falciforme/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Imunodeficiência Combinada Severa/terapia , Talassemia beta/terapia , Anemia Falciforme/genética , Animais , Engenharia Genética , Humanos , Imunodeficiência Combinada Severa/genética , Talassemia beta/genéticaRESUMO
ARHGEF1 is a RhoA-specific guanine nucleotide exchange factor expressed in hematopoietic cells. We used whole-exome sequencing to identify compound heterozygous mutations in ARHGEF1, resulting in the loss of ARHGEF1 protein expression in 2 primary antibody-deficient siblings presenting with recurrent severe respiratory tract infections and bronchiectasis. Both ARHGEF1-deficient patients showed an abnormal B cell immunophenotype, with a deficiency in marginal zone and memory B cells and an increased frequency of transitional B cells. Furthermore, the patients' blood contained immature myeloid cells. Analysis of a mediastinal lymph node from one patient highlighted the small size of the germinal centers and an abnormally high plasma cell content. On the molecular level, T and B lymphocytes from both patients displayed low RhoA activity and low steady-state actin polymerization (even after stimulation of lysophospholipid receptors). As a consequence of disturbed regulation of the RhoA downstream target Rho-associated kinase I/II (ROCK), the patients' lymphocytes failed to efficiently restrain AKT phosphorylation. Enforced ARHGEF1 expression or drug-induced activation of RhoA in the patients' cells corrected the impaired actin polymerization and AKT regulation. Our results indicate that ARHGEF1 activity in human lymphocytes is involved in controlling actin cytoskeleton dynamics, restraining PI3K/AKT signaling, and confining B lymphocytes and myelocytes within their dedicated functional environment.
Assuntos
Linfócitos B , Doenças da Imunodeficiência Primária , Transdução de Sinais , Linfócitos B/imunologia , Linfócitos B/patologia , Feminino , Centro Germinativo/imunologia , Centro Germinativo/patologia , Humanos , Memória Imunológica/genética , Masculino , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/imunologia , Doenças da Imunodeficiência Primária/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Fatores de Troca de Nucleotídeo Guanina Rho/deficiência , Fatores de Troca de Nucleotídeo Guanina Rho/imunologia , Irmãos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Quinases Associadas a rho/genética , Quinases Associadas a rho/imunologia , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Autosomal dominant gain-of-function mutations in human stimulator of interferon genes (STING) lead to a severe autoinflammatory disease called STING-associated vasculopathy with onset in infancy that is associated with enhanced expression of interferon-stimulated gene transcripts. OBJECTIVE: The goal of this study was to analyze the phenotype of a new mouse model of STING hyperactivation and the role of type I interferons in this system. METHODS: We generated a knock-in model carrying an amino acid substitution (V154M) in mouse STING, corresponding to a recurrent mutation seen in human patients with STING-associated vasculopathy with onset in infancy. Hematopoietic development and tissue histology were analyzed. Lymphocyte activation and proliferation were assessed in vitro. STING V154M/wild-type (WT) mice were crossed to IFN-α/ß receptor (IFNAR) knockout mice to evaluate the type I interferon dependence of the mutant Sting phenotype recorded. RESULTS: In STING V154M/WT mice we detected variable expression of inflammatory infiltrates in the lungs and kidneys. These mice showed a marked decrease in survival and developed a severe combined immunodeficiency disease (SCID) affecting B, T, and natural killer cells, with an almost complete lack of antibodies and a significant expansion of monocytes and granulocytes. The blockade in B- and T-cell development was present from early immature stages in bone marrow and thymus. In addition, in vitro experiments revealed an intrinsic proliferative defect of mature T cells. Although the V154M/WT mutant demonstrated increased expression of interferon-stimulated genes, the SCID phenotype was not reversed in STING V154M/WT IFNAR knockout mice. However, the antiproliferative defect in T cells was rescued partially by IFNAR deficiency. CONCLUSIONS: STING gain-of-function mice developed an interferon-independent SCID phenotype with a T-cell, B-cell, and natural killer cell developmental defect and hypogammaglobulinemia that is associated with signs of inflammation in lungs and kidneys. Only the intrinsic proliferative defect of T cells was partially interferon dependent.
Assuntos
Linfócitos B/fisiologia , Inflamação/genética , Células Matadoras Naturais/imunologia , Proteínas de Membrana/genética , Mutação/genética , Imunodeficiência Combinada Severa/genética , Linfócitos T/fisiologia , Agamaglobulinemia , Animais , Diferenciação Celular/genética , Modelos Animais de Doenças , Humanos , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/genéticaRESUMO
Deletion of chromosome 6q is a well-recognized abnormality found in poor-prognosis T-cell acute lymphoblastic leukemia (T-ALL). Using integrated genomic approaches, we identified two candidate haploinsufficient genes contiguous at 6q14, SYNCRIP (encoding hnRNP-Q) and SNHG5 (that hosts snoRNAs), both involved in regulating RNA maturation and translation. Combined silencing of both genes, but not of either gene alone, accelerated leukemogeneis in a Tal1/Lmo1/Notch1-driven mouse model, demonstrating the tumor-suppressive nature of the two-gene region. Proteomic and translational profiling of cells in which we engineered a short 6q deletion by CRISPR/Cas9 genome editing indicated decreased ribosome and mitochondrial activities, suggesting that the resulting metabolic changes may regulate tumor progression. Indeed, xenograft experiments showed an increased leukemia-initiating cell activity of primary human leukemic cells upon coextinction of SYNCRIP and SNHG5. Our findings not only elucidate the nature of 6q deletion but also highlight the role of ribosomes and mitochondria in T-ALL tumor progression. SIGNIFICANCE: The oncogenic role of 6q deletion in T-ALL has remained elusive since this chromosomal abnormality was first identified more than 40 years ago. We combined genomic analysis and functional models to show that the codeletion of two contiguous genes at 6q14 enhances malignancy through deregulation of a ribosome-mitochondria axis, suggesting the potential for therapeutic intervention.This article is highlighted in the In This Issue feature, p. 1494.
Assuntos
Deleção Cromossômica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Leucemia de Células T/genética , RNA Longo não Codificante/genética , Ribossomos/metabolismo , Animais , Linhagem Celular Tumoral , Cromossomos Humanos Par 6 , Progressão da Doença , Perfilação da Expressão Gênica , Haploinsuficiência , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Interferência de RNA , RNA Longo não Codificante/metabolismo , Transplante HeterólogoRESUMO
Lentiviral vectors have emerged as an efficient, safe therapeutic tool for gene therapy based on hematopoietic stem cells (HSCs) or T cells. However, the monitoring of transduced cells in preclinical models remains challenging because of the inefficient transduction of murine primary T cells with lentiviral vectors, in contrast to gammaretroviral vectors. The use of this later in preclinical proof of concept is not considered as relevant when a lentiviral vector will be used in a clinical trial. Hence, there is an urgent need to develop an efficient transduction protocol for murine cells with lentiviral vectors. Here, we describe an optimized protocol in which a nontoxic transduction enhancer (Lentiboost) enables the efficient transduction of primary murine T cells with lentiviral vectors. The optimized protocol combines low toxicity and high transduction efficiency. We achieved a high-level transduction of murine CD4+ and CD8+ T cells with a VSV-G-pseudotyped lentiviral vector with no changes in the phenotypes of transduced T cells, which were stable and long-lived in culture. This enhancer also increased the transduction of murine HSCs. Hence, use of this new transduction enhancer overcomes the limitations of lentiviral vectors in preclinical experiments and should facilitate the translation of strategies based on lentiviral vectors from the bench to the clinic.
RESUMO
Autologous transplantation of hematopoietic stem cells transduced with a lentiviral vector (LV) expressing an anti-sickling HBB variant is a potential treatment for sickle cell disease (SCD). With a clinical trial as our ultimate goal, we generated LV constructs containing an anti-sickling HBB transgene (HBBAS3), a minimal HBB promoter, and different combinations of DNase I hypersensitive sites (HSs) from the locus control region (LCR). Hematopoietic stem progenitor cells (HSPCs) from SCD patients were transduced with LVs containing either HS2 and HS3 (ß-AS3) or HS2, HS3, and HS4 (ß-AS3 HS4). The inclusion of the HS4 element drastically reduced vector titer and infectivity in HSPCs, with negligible improvement of transgene expression. Conversely, the LV containing only HS2 and HS3 was able to efficiently transduce SCD bone marrow and Plerixafor-mobilized HSPCs, with anti-sickling HBB representing up to â¼60% of the total HBB-like chains. The expression of the anti-sickling HBB and the reduced incorporation of the ßS-chain in hemoglobin tetramers allowed up to 50% reduction in the frequency of RBC sickling under hypoxic conditions. Together, these results demonstrate the ability of a high-titer LV to express elevated levels of a potent anti-sickling HBB transgene ameliorating the SCD cell phenotype.
RESUMO
We report on 4 children who presented with aseptic panniculitis associated with inherited immunodeficiency. Three patients had a B-cell immunodeficiency resulting from mutations in the TRNT1 and NF-κb2 genes (no mutation was found in the third patient), and 1 had a T-cell deficiency (mutation in the LCK gene). Panniculitis occurred before the age of 2 years in the 4 patients and preceded the onset of recurrent infections because of immunodeficiency in 2. It presented either as nodules, which resolved spontaneously within 1 to 2 weeks (3 patients), or chronic ulcerative lesions (1 patient) associated with unexplained fever and elevated acute phase reactants, without evidence of infection or high-titer autoantibodies. Febrile nodules relapsed in 2 patients, and recurrent attacks of unexplained fever (without relapse of panniculitis) occurred in the third. Skin biopsy revealed predominantly lympho-histiocytic or septal neutrophilic panniculitis in 1 and 3 patients, respectively. Panniculitis was associated with dermal involvement in the 4 patients. Patients with B-cell deficiency received monthly intravenous immunoglobulin replacement. Two patients who underwent bone marrow transplant died of bone marrow transplant-related complications. The 2 remaining patients had persistent, mild autoinflammatory disease, which did not require specific treatment. In these cases, the need for careful immunologic evaluation of patients who present with unexplained panniculitis, especially early-onset panniculitis before the age of 2 years, is highlighted.
Assuntos
Síndromes de Imunodeficiência/complicações , Síndromes de Imunodeficiência/genética , Paniculite/complicações , Paniculite/imunologia , Linfócitos B/imunologia , Transplante de Medula Óssea/efeitos adversos , Pré-Escolar , Feminino , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Síndromes de Imunodeficiência/terapia , Lactente , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Masculino , Mutação , Subunidade p52 de NF-kappa B/genética , Nucleotidiltransferases/genética , Complicações Pós-Operatórias , Linfócitos T/imunologiaRESUMO
Sickle cell disease is characterized by chronic anemia and vaso-occlusive crises, which eventually lead to multi-organ damage and premature death. Hematopoietic stem cell transplantation is the only curative treatment but it is limited by toxicity and poor availability of HLA-compatible donors. A gene therapy approach based on the autologous transplantation of lentiviral-corrected hematopoietic stem and progenitor cells was shown to be efficacious in one patient. However, alterations of the bone marrow environment and properties of the red blood cells hamper the harvesting and immunoselection of patients' stem cells from bone marrow. The use of Filgrastim to mobilize large numbers of hematopoietic stem and progenitor cells into the circulation has been associated with severe adverse events in sickle cell patients. Thus, broader application of the gene therapy approach requires the development of alternative mobilization methods. We set up a phase I/II clinical trial whose primary objective was to assess the safety of a single injection of Plerixafor in sickle cell patients undergoing red blood cell exchange to decrease the hemoglobin S level to below 30%. The secondary objective was to measure the efficiency of mobilization and isolation of hematopoietic stem and progenitor cells. No adverse events were observed. Large numbers of CD34+ cells were mobilized extremely quickly. Importantly, the mobilized cells contained high numbers of hematopoietic stem cells, expressed high levels of stemness genes, and engrafted very efficiently in immunodeficient mice. Thus, Plerixafor can be safely used to mobilize hematopoietic stem cells in sickle cell patients; this finding opens up new avenues for treatment approaches based on gene addition and genome editing. Clinicaltrials.gov identifier: NCT02212535.
Assuntos
Anemia Falciforme/terapia , Transfusão de Sangue , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Compostos Heterocíclicos/administração & dosagem , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Fármacos Anti-HIV/administração & dosagem , Antígenos CD34/metabolismo , Antidrepanocíticos/administração & dosagem , Benzilaminas , Estudos de Casos e Controles , Células Cultivadas , Estudos de Coortes , Ciclamos , Células-Tronco Hematopoéticas/citologia , Humanos , Hidroxiureia/administração & dosagemRESUMO
BACKGROUND: Common variable immunodeficiency (CVID) is characterized by infections and hypogammaglobulinemia. Neutropenia is rare during CVID. METHODS: The French DEFI study enrolled patients with primary hypogammaglobulinemia. Patients with CVID and neutropenia were retrospectively analyzed. RESULTS: Among 473 patients with CVID, 16 patients displayed neutropenia (lowest count [0-1400]*106/L). Sex ratio (M/F) was 10/6. Five patients died during the follow-up (11 years) with an increased percentage of deaths compared to the whole DEFI group (31.3 vs 3.4%, P < 0.05). Neutropenia was diagnosed for 10 patients before 22 years old. The most frequent symptoms, except infections, were autoimmune cytopenia, i.e., thrombopenia or anemia (11/16). Ten patients were affected with lymphoproliferative diseases. Two patients were in the infection only group and the others belonged to one or several other CVID groups. The median level of IgG was 2.6 g/L [0.35-4.4]. Most patients presented increased numbers of CD21low CD38low B cell, as already described in CVID autoimmune cytopenia group. Neutropenia was considered autoimmune in 11 cases. NGS for 52 genes of interest was performed on 8 patients. No deleterious mutations were found in LRBA, CTLA4, and PIK3. More than one potentially damaging variant in other genes associated with CVID were present in most patients arguing for a multigene process. CONCLUSION: Neutropenia is generally associated with another cytopenia and presumably of autoimmune origin during CVID. In the DEFI study, neutropenia is coupled with more severe clinical outcomes. It appears as an "alarm bell" considering patients' presentation and the high rate of deaths. Whole exome sequencing diagnosis should improve management.
Assuntos
Imunodeficiência de Variável Comum/epidemiologia , Neutropenia/epidemiologia , Adolescente , Adulto , Criança , Pré-Escolar , Imunodeficiência de Variável Comum/sangue , Imunodeficiência de Variável Comum/genética , Imunodeficiência de Variável Comum/imunologia , Comorbidade , Feminino , França/epidemiologia , Humanos , Imunoglobulinas/sangue , Lactente , Recém-Nascido , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Neutropenia/sangue , Neutropenia/genética , Neutropenia/imunologia , Sequenciamento do Exoma , Adulto JovemRESUMO
When considering inherited diseases that can be treated by gene transfer into hematopoietic stem cells (HSCs), there are only two in which the HSC and progenitor cell distribution inside the bone marrow and its microenvironment are exactly the same as in a healthy subject: metachromatic leukodystrophy (MLD) and adrenoleukodystrophy (ALD). In all other settings [X-linked severe combined immunodeficiency (X-SCID), adenosine deaminase deficiency, Wiskott-Aldrich syndrome, and ß-hemoglobinopathies], the bone marrow content of the different stem and precursor cells and the cells' relationship with the stroma have very specific characteristics. These peculiarities can influence the cells' harvesting and behavior in culture, and the postgraft uptake and further behavior of the gene-modified hematopoietic/precursor cells. In the present mini-review, we shall briefly summarize these characteristics and outline the possible consequences and challenges.
Assuntos
Doenças da Medula Óssea/terapia , Medula Óssea/patologia , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Animais , Doenças da Medula Óssea/patologia , HumanosRESUMO
Patients with mutations in the UNC13D gene (coding for Munc13-4 protein) suffer from familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a life-threatening immune and hyperinflammatory disorder. The only curative treatment is allogeneic hematopoietic stem cell (HSC) transplantation, although the posttreatment survival rate is not satisfactory. Here, we demonstrate the curative potential of UNC13D gene correction of HSCs in a murine model of FHL3. We generated a self-inactivating lentiviral vector, used it to complement HSCs from Unc13d-deficient (Jinx) mice, and transplanted the cells back into the irradiated Jinx recipients. This procedure led to complete reconstitution of the immune system (ie, to wild-type levels). The recipients were then challenged with lymphocytic choriomeningitis virus to induce hemophagocytic lymphohistiocytosis (HLH)-like manifestations. All the clinical and biological signs of HLH were significantly reduced in mice having undergone HSC UNC13D gene correction than in nontreated animals. This beneficial effect was evidenced by the correction of blood cytopenia, body weight gain, normalization of the body temperature, decreased serum interferon-γ level, recovery of liver damage, and decreased viral load. These improvements can be explained by the restoration of the CD8+ T lymphocytes' cytotoxic function (as demonstrated here in an in vitro degranulation assay). Overall, our results demonstrate the efficacy of HSC gene therapy in an FHL-like setting of immune dysregulation.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4/imunologia , Linfo-Histiocitose Hemofagocítica , Transtornos Linfoproliferativos , Proteínas de Membrana/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/patologia , Terapia Genética , Herpesvirus Humano 4/genética , Humanos , Linfo-Histiocitose Hemofagocítica/genética , Linfo-Histiocitose Hemofagocítica/imunologia , Linfo-Histiocitose Hemofagocítica/patologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/patologia , Transtornos Linfoproliferativos/virologia , Proteínas de Membrana/deficiência , Camundongos , Camundongos SCID , Transdução GenéticaRESUMO
The nature of gut intraepithelial lymphocytes (IELs) lacking antigen receptors remains controversial. Herein we showed that, in humans and in mice, innate intestinal IELs expressing intracellular CD3 (iCD3(+)) differentiate along an Id2 transcription factor (TF)-independent pathway in response to TF NOTCH1, interleukin-15 (IL-15), and Granzyme B signals. In NOTCH1-activated human hematopoietic precursors, IL-15 induced Granzyme B, which cleaved NOTCH1 into a peptide lacking transcriptional activity. As a result, NOTCH1 target genes indispensable for T cell differentiation were silenced and precursors were reprogrammed into innate cells with T cell marks including intracellular CD3 and T cell rearrangements. In the intraepithelial lymphoma complicating celiac disease, iCD3(+) innate IELs acquired gain-of-function mutations in Janus kinase 1 or Signal transducer and activator of transcription 3, which enhanced their response to IL-15. Overall we characterized gut T cell-like innate IELs, deciphered their pathway of differentiation and showed their malignant transformation in celiac disease.
Assuntos
Doença Celíaca/imunologia , Interleucina-15/imunologia , Intestinos/imunologia , Linfoma/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Complexo CD3/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Granzimas/imunologia , Humanos , Proteína 2 Inibidora de Diferenciação/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor Notch1/imunologia , Fator de Transcrição STAT3/imunologia , Transdução de Sinais/imunologia , Transcrição Gênica/imunologiaRESUMO
Lymphoid-committed CD34(+)lin(-)CD10(+)CD24(-) progenitors undergo a rebound at month 3 after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the absence of acute graft-versus-host disease (aGVHD). Here, we analyzed transcriptional programs of cell-sorted circulating lymphoid-committed progenitors and CD34(+)Lin(-)CD10(-) nonlymphoid progenitors in 11 allo-HSCT patients who had (n = 5) or had not (n = 6) developed grade 2 or 3 aGVHD and in 7 age-matched healthy donors. Major upregulated pathways include protein synthesis, energy production, cell cycle regulation, and cytoskeleton organization. Notably, genes from protein biogenesis, translation machinery, and cell cycle (CDK6) were overexpressed in progenitors from patients in the absence of aGVHD compared with healthy donors and patients affected by aGVHD. Expression of many genes from the mitochondrial oxidative phosphorylation metabolic pathway leading to ATP production were more specifically increased in lymphoid-committed progenitors in the absence of aGVHD. This was also the case for genes involved in cell mobilization such as those regulating Rho GTPase activity. In all, we found that circulating lymphoid-committed progenitors undergo profound changes in metabolism, favoring cell proliferation, energy production, and cell mobilization after allo-HSCT in humans. These mechanisms are abolished in the case of aGVHD or its treatment, indicating a persistent cell-intrinsic defect after exit from the bone marrow.
Assuntos
Metabolismo Energético , Transplante de Células-Tronco Hematopoéticas , Células Progenitoras Linfoides/metabolismo , Adulto , Biomarcadores , Feminino , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Fenótipo , Transplante Homólogo , Adulto JovemRESUMO
More than 20 years ago, X-linked severe combined immunodeficiency (SCID-X1) appeared to be the best condition to test the feasibility of hematopoietic stem cell gene therapy. The seminal SCID-X1 clinical studies, based on first-generation gammaretroviral vectors, demonstrated good long-term immune reconstitution in most treated patients despite the occurrence of vector-related leukemia in a few of them. This gene therapy has successfully enabled correction of the T cell defect. Natural killer and B cell defects were only partially restored, most likely due to the absence of a conditioning regimen. The success of these pioneering trials paved the way for the extension of gene-based treatment to many other diseases of the hematopoietic system, but the unfortunate serious adverse events led to extensive investigations to define the retrovirus integration profiles. This review puts into perspective the clinical experience of gene therapy for SCID-X1, with the development and implementation of new generations of safer vectors such as self-inactivating gammaretroviral or lentiviral vectors as well as major advances in integrome knowledge.
Assuntos
Gammaretrovirus/genética , Terapia Genética/métodos , Vetores Genéticos/química , Lentivirus/genética , Retroviridae/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Linfócitos B/imunologia , Linfócitos B/patologia , Ensaios Clínicos como Assunto , Gammaretrovirus/imunologia , Vetores Genéticos/imunologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Lentivirus/imunologia , Segurança do Paciente , Retroviridae/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Integração Viral/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/patologiaRESUMO
BACKGROUND: Myb-Like, SWIRM, and MPN domains 1 (MYSM1) is a metalloprotease that deubiquitinates the K119-monoubiquitinated form of histone 2A (H2A), a chromatin marker associated with gene transcription silencing. Likewise, it has been reported that murine Mysm1 participates in transcription derepression of genes, among which are transcription factors involved in hematopoietic stem cell homeostasis, hematopoiesis, and lymphocyte differentiation. However, whether MYSM1 has a similar function in human subjects remains unclear. Here we describe a patient presenting with a complete lack of B lymphocytes, T-cell lymphopenia, defective hematopoiesis, and developmental abnormalities. OBJECTIVES: We sought to characterize the underlying genetic cause of this syndrome. METHODS: We performed genome-wide homozygosity mapping, followed by whole-exome sequencing. RESULTS: Genetic analysis revealed that this novel disorder is caused by a homozygous MYSM1 missense mutation affecting the catalytic site within the deubiquitinase JAB1/MPN/Mov34 (JAMM)/MPN domain. Remarkably, during the course of our study, the patient recovered a normal immunohematologic phenotype. Genetic analysis indicated that this improvement originated from a spontaneous genetic reversion of the MYSM1 mutation in a hematopoietic stem cell. CONCLUSIONS: We here define a novel human immunodeficiency and provide evidence that MYSM1 is essential for proper immunohematopoietic development in human subjects. In addition, we describe one of the few examples of spontaneous in vivo genetic cure of a human immunodeficiency.
Assuntos
Proteínas de Ligação a DNA/genética , Síndromes de Imunodeficiência/genética , Fatores de Transcrição/genética , Linfócitos B/citologia , Diferenciação Celular , Hematopoese/genética , Humanos , Lactente , Linfopenia/genética , Masculino , Mutação , Linfócitos T/citologia , Transativadores , Proteases Específicas de UbiquitinaRESUMO
OBJECTIVES: Zidovudine and tenofovir are the two main nucleos(t)ide analogs used to prevent mother-to-child transmission of HIV. In vitro, both drugs bind to and integrate into human DNA and inhibit telomerase. The objective of the present study was to assess the genotoxic effects of either zidovudine or tenofovir-based combination therapies on cord blood cells in newborns exposed in utero. DESIGN: We compared the aneuploid rate and the gene expression profiles in cord blood samples from newborns exposed either to zidovudine or tenofovir-based combination therapies during pregnancy and from unexposed controls (nâ=â8, 9, and 8, respectively). METHODS: The aneuploidy rate was measured on the cord blood T-cell karyotype. Gene expression profiles of cord blood T cells and hematopoietic stem and progenitor cells were determined with microarrays, analyzed in a gene set enrichment analysis and confirmed by real-time quantitative PCRs. RESULTS: Aneuploidy was more frequent in the zidovudine-exposed group (26.3%) than in the tenofovir-exposed group (14.2%) or in controls (13.3%; Pâ<â0.05 for both). The transcription of genes involved in DNA repair, telomere maintenance, nucleotide metabolism, DNA/RNA synthesis, and the cell cycle was deregulated in samples from both the zidovudine and the tenofovir-exposed groups. CONCLUSION: Although tenofovir has a lower clastogenic impact than zidovudine, gene expression profiling showed that both drugs alter the transcription of DNA repair and telomere maintenance genes.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Sangue Fetal/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Complicações Infecciosas na Gravidez/tratamento farmacológico , Tenofovir/uso terapêutico , Zidovudina/uso terapêutico , Aneuploidia , Feminino , Expressão Gênica , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/transmissão , Humanos , Recém-Nascido , Troca Materno-Fetal , Gravidez , Complicações Infecciosas na Gravidez/virologiaRESUMO
V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rendering RAG1/2 targeting a potentially important regulator of lymphoid differentiation. We show that within the human TCR-α/δ locus, Dδ2-Dδ3 rearrangements occur at a very immature thymic, CD34(+)/CD1a(-)/CD7(+dim) stage, before Dδ2(Dδ3)-Jδ1 rearrangements. These strictly ordered rearrangements are regulated by mechanisms acting beyond chromatin accessibility. Importantly, direct Dδ2-Jδ1 rearrangements are prohibited by a B12/23 restriction and ordered human TCR-δ gene assembly requires RUNX1 protein, which binds to the Dδ2-23RSS, interacts with RAG1, and enhances RAG1 deposition at this site. This RUNX1-mediated V(D)J recombinase targeting imposes the use of two Dδ gene segments in human TCR-δ chains. Absence of this RUNX1 binding site in the homologous mouse Dδ1-23RSS provides a molecular explanation for the lack of ordered TCR-δ gene assembly in mice and may underlie differences in early lymphoid differentiation between these species.