RESUMEN
Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools.
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Mielopoyesis , Neutrófilos , Biomarcadores/metabolismo , Humanos , Interferones/genética , Interferones/metabolismo , Neutrófilos/metabolismo , Plásticos/metabolismoRESUMEN
ABSTRACT: In physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPCs) are present in the peripheral blood, but their contribution to human hematopoiesis remain unsolved. By integrating advanced immunophenotyping, single-cell transcriptional and functional profiling, and integration site (IS) clonal tracking, we unveiled the biological properties and the transcriptional features of human cHSPC subpopulations in relationship to their bone marrow (BM) counterpart. We found that cHSPCs reduced in cell count over aging and are enriched for primitive, lymphoid, and erythroid subpopulations, showing preactivated transcriptional and functional state. Moreover, cHSPCs have low expression of multiple BM-retention molecules but maintain their homing potential after xenotransplantation. By generating a comprehensive human organ-resident HSPC data set based on single-cell RNA sequencing data, we detected organ-specific seeding properties of the distinct trafficking HSPC subpopulations. Notably, circulating multi-lymphoid progenitors are primed for seeding the thymus and actively contribute to T-cell production. Human clonal tracking data from patients receiving gene therapy (GT) also showed that cHSPCs connect distant BM niches and participate in steady-state hematopoietic production, with primitive cHSPCs having the highest recirculation capability to travel in and out of the BM. Finally, in case of hematopoietic impairment, cHSPCs composition reflects the BM-HSPC content and might represent a biomarker of the BM state for clinical and research purposes. Overall, our comprehensive work unveiled fundamental insights into the in vivo dynamics of human HSPC trafficking and its role in sustaining hematopoietic homeostasis. GT patients' clinical trials were registered at ClinicalTrials.gov (NCT01515462 and NCT03837483) and EudraCT (2009-017346-32 and 2018-003842-18).
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Hematopoyesis , Células Madre Hematopoyéticas , Homeostasis , Animales , Humanos , Ratones , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Análisis de la Célula IndividualRESUMEN
Longitudinal clonal tracking studies based on high-throughput sequencing technologies supported safety and long-term efficacy and unraveled hematopoietic reconstitution in many gene therapy applications with unprecedented resolution. However, monitoring patients over a decade-long follow-up entails a constant increase of large data volume with the emergence of critical computational challenges, unfortunately not addressed by currently available tools. Here we present ISAnalytics, a new R package for comprehensive and high-throughput clonal tracking studies using vector integration sites as markers of cellular identity. Once identified the clones externally from ISAnalytics and imported in the package, a wide range of implemented functionalities are available to users for assessing the safety and long-term efficacy of the treatment, here described in a clinical trial use case for Hurler disease, and for supporting hematopoietic stem cell biology in vivo with longitudinal analysis of clones over time, proliferation and differentiation. ISAnalytics is conceived to be metadata-driven, enabling users to focus on biological questions and hypotheses rather than on computational aspects. ISAnalytics can be fully integrated within laboratory workflows and standard procedures. Moreover, ISAnalytics is designed with efficient and scalable data structures, benchmarked with previous methods, and grants reproducibility and full analytical control through interactive web-reports and a module with Shiny interface. The implemented functionalities are flexible for all viral vector-based clonal tracking applications as well as genetic barcoding or cancer immunotherapies.
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Terapia Genética , Células Madre Hematopoyéticas , Humanos , Células Clonales , Terapia Genética/efectos adversos , Secuenciación de Nucleótidos de Alto Rendimiento , Reproducibilidad de los Resultados , Ensayos Clínicos como AsuntoRESUMEN
BACKGROUND: Allogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant [MPSIH]). However, this treatment is only partially curative and is associated with complications. METHODS: We are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an α-L-iduronidase (IDUA)-encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years. RESULTS: We now report interim results. The children's mean (±SD) age at the time of HSPC gene therapy was 1.9±0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced joint stiffness, and normal growth in line with World Health Organization growth charts. CONCLUSIONS: The delivery of HSPC gene therapy in patients with MPSIH resulted in extensive metabolic correction in peripheral tissues and the central nervous system. (Funded by Fondazione Telethon and others; ClinicalTrials.gov number, NCT03488394; EudraCT number, 2017-002430-23.).
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Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Iduronidasa/metabolismo , Mucopolisacaridosis I/terapia , Preescolar , Femenino , Estudios de Seguimiento , Vectores Genéticos , Glicosaminoglicanos/orina , Humanos , Iduronidasa/deficiencia , Iduronidasa/genética , Lactante , Lentivirus , Masculino , Mucopolisacaridosis I/metabolismo , Mutación , Trasplante de Células Madre , Trasplante AutólogoRESUMEN
Acute myeloid leukaemia (AML) relapse after allogeneic haematopoietic cell transplantation (allo-HCT) is often driven by immune-related mechanisms and associated with poor prognosis. Immune checkpoint inhibitors combined with hypomethylating agents (HMA) may restore or enhance the graft-versus-leukaemia effect. Still, data about using this combination regimen after allo-HCT are limited. We conducted a prospective, phase II, open-label, single-arm study in which we treated patients with haematological AML relapse after allo-HCT with HMA plus the anti-PD-1 antibody nivolumab. The response was correlated with DNA-, RNA- and protein-based single-cell technology assessments to identify biomarkers associated with therapeutic efficacy. Sixteen patients received a median number of 2 (range 1-7) nivolumab applications. The overall response rate (CR/PR) at day 42 was 25%, and another 25% of the patients achieved stable disease. The median overall survival was 15.6 months. High-parametric cytometry documented a higher frequency of activated (ICOS+ , HLA-DR+ ), low senescence (KLRG1- , CD57- ) CD8+ effector T cells in responders. We confirmed these findings in a preclinical model. Single-cell transcriptomics revealed a pro-inflammatory rewiring of the expression profile of T and myeloid cells in responders. In summary, the study indicates that the post-allo-HCT HMA/nivolumab combination induces anti-AML immune responses in selected patients and could be considered as a bridging approach to a second allo-HCT. Trial-registration: EudraCT-No. 2017-002194-18.
RESUMEN
We developed a brain and spine magnetic resonance scoring system based on a magnetic resonance assessment of 9 patients with mucopolysaccharidosis type I-Hurler who underwent hematopoietic stem-cell transplantation. The score is reliable and correlates with long-term clinical and cognitive outcome in patients with mucopolysaccharidosis type I-Hurler.
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Encéfalo/diagnóstico por imagen , Mucopolisacaridosis I/diagnóstico por imagen , Médula Espinal/diagnóstico por imagen , Adolescente , Encéfalo/patología , Niño , Preescolar , Femenino , Estudios de Seguimiento , Trasplante de Células Madre Hematopoyéticas , Humanos , Imagen por Resonancia Magnética , Masculino , Mucopolisacaridosis I/patología , Mucopolisacaridosis I/cirugía , Neuroimagen , Estudios Retrospectivos , Médula Espinal/patologíaRESUMEN
Tumor progression is accompanied by an altered myelopoiesis causing the accumulation of immunosuppressive cells. Here, we showed that miR-142-3p downregulation promoted macrophage differentiation and determined the acquisition of their immunosuppressive function in tumor. Tumor-released cytokines signaling through gp130, the common subunit of the interleukin-6 cytokine receptor family, induced the LAP∗ isoform of C/EBPß transcription factor, promoting macrophage generation. miR-142-3p downregulated gp130 by canonical binding to its messenger RNA (mRNA) 3' UTR and repressed C/EBPß LAP∗ by noncanonical binding to its 5' mRNA coding sequence. Enforced miR expression impaired macrophage differentiation both in vitro and in vivo. Mice constitutively expressing miR-142-3p in the bone marrow showed a marked increase in survival following immunotherapy with tumor-specific T lymphocytes. By modulating a specific miR in bone marrow precursors, we thus demonstrated the feasibility of altering tumor-induced macrophage differentiation as a potent tool to improve the efficacy of cancer immunotherapy.
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Inmunoterapia/métodos , Macrófagos/inmunología , MicroARNs/metabolismo , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , ARN Mensajero/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Receptor gp130 de Citocinas/metabolismo , Inmunoterapia/tendencias , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , Mielopoyesis/genética , Neoplasias Experimentales/terapia , ARN Mensajero/genética , Transducción de Señal , Esteroide Isomerasas/genética , Esteroide Isomerasas/metabolismo , Linfocitos T/inmunología , Linfocitos T/trasplante , Transgenes/genética , Escape del TumorRESUMEN
Chronic granulomatous disease (CGD) is a rare inherited disorder due to loss-of-function mutations in genes encoding the NADPH oxidase subunits. Hematopoietic stem and progenitor cell (HSPC) gene therapy (GT) using regulated lentiviral vectors (LVs) has emerged as a promising therapeutic option for CGD patients. We performed non-clinical Good Laboratory Practice (GLP) and laboratory-grade studies to assess the safety and genotoxicity of LV targeting myeloid-specific Gp91phox expression in X-linked chronic granulomatous disease (XCGD) mice. We found persistence of gene-corrected cells for up to 1 year, restoration of Gp91phox expression and NADPH oxidase activity in XCGD phagocytes, and reduced tissue inflammation after LV-mediated HSPC GT. Although most of the mice showed no hematological or biochemical toxicity, a small subset of XCGD GT mice developed T cell lymphoblastic lymphoma (2.94%) and myeloid leukemia (5.88%). No hematological malignancies were identified in C57BL/6 mice transplanted with transduced XCGD HSPCs. Integration pattern analysis revealed an oligoclonal composition with rare dominant clones harboring vector insertions near oncogenes in mice with tumors. Collectively, our data support the long-term efficacy of LV-mediated HSPC GT in XCGD mice and provide a safety warning because the chronic inflammatory XCGD background may contribute to oncogenesis.
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Terapia Genética , Vectores Genéticos/genética , Enfermedad Granulomatosa Crónica/complicaciones , Enfermedad Granulomatosa Crónica/terapia , Neoplasias Hematológicas/etiología , Lentivirus/genética , Animales , Modelos Animales de Enfermedad , Terapia Genética/efectos adversos , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Enfermedad Granulomatosa Crónica/genética , Humanos , Ratones , NADPH Oxidasa 2/genética , NADPH Oxidasa 2/metabolismo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.
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Trasplante de Células Madre Hematopoyéticas , Osteopetrosis , ATPasas de Translocación de Protón Vacuolares , Antígenos CD34 , Terapia Genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Osteoclastos/metabolismo , Osteopetrosis/genética , Osteopetrosis/terapia , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
ETV6-RUNX1 (E/R) fusion gene, arising in utero from translocation t(12;21)(p13:q22), is the most frequent alteration in childhood acute lymphoblastic leukemia (ALL). However, E/R is insufficient to cause overt leukemia since it generates a clinically silent pre-leukemic clone which persists in the bone marrow but fails to out-compete normal progenitors. Conversely, pre-leukemic cells show increased susceptibility to transformation following additional genetic insults. Infections/inflammation are the most accredited triggers for mutations accumulation and leukemic transformation in E/R+ pre-leukemic cells. However, precisely how E/R and inflammation interact in promoting leukemia is still poorly understood. Here we demonstrate that IL6/TNFα/ILß pro-inflammatory cytokines cooperate with BM-MSC in promoting the emergence of E/R+ Ba/F3 over their normal counterparts by differentially affecting their proliferation and survival. Moreover, IL6/TNFα/ILß-stimulated BM-MSC strongly attract E/R+ Ba/F3 in a CXCR2-dependent manner. Interestingly, E/R-expressing human CD34+ IL7R+ progenitors, a putative population for leukemia initiation during development, were preserved in the presence of BM-MSC and IL6/TNFα/ILß compared to their normal counterparts. Finally, the extent of DNA damage increases within the inflamed niche in both control and E/R-expressing Ba/F3, potentially leading to transformation in the apoptosis-resistant pre-leukemic clone. Overall, our data provide new mechanistic insights into childhood ALL pathogenesis.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Citocinas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Humanos , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Translocación GenéticaRESUMEN
Mendelian susceptibility to mycobacterial disease is a rare primary immunodeficiency characterized by severe infections caused by weakly virulent mycobacteria. Biallelic null mutations in genes encoding interferon gamma receptor 1 or 2 (IFNGR1 or IFNGR2) result in a life-threatening disease phenotype in early childhood. Recombinant interferon γ (IFN-γ) therapy is inefficient, and hematopoietic stem cell transplantation has a poor prognosis. Thus, we developed a hematopoietic stem cell (HSC) gene therapy approach using lentiviral vectors that express Ifnγr1 either constitutively or myeloid specifically. Transduction of mouse Ifnγr1-/- HSCs led to stable IFNγR1 expression on macrophages, which rescued their cellular responses to IFN-γ. As a consequence, genetically corrected HSC-derived macrophages were able to suppress T-cell activation and showed restored antimycobacterial activity against Mycobacterium avium and Mycobacterium bovis Bacille Calmette-Guérin (BCG) in vitro. Transplantation of genetically corrected HSCs into Ifnγr1-/- mice before BCG infection prevented manifestations of severe BCG disease and maintained lung and spleen organ integrity, which was accompanied by a reduced mycobacterial burden in lung and spleen and a prolonged overall survival in animals that received a transplant. In summary, we demonstrate an HSC-based gene therapy approach for IFNγR1 deficiency, which protects mice from severe mycobacterial infections, thereby laying the foundation for a new therapeutic intervention in corresponding human patients.
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Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Infecciones por Mycobacterium/prevención & control , Sustancias Protectoras , Receptores de Interferón/genética , Animales , Células Cultivadas , Trasplante de Células Madre Hematopoyéticas/métodos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium avium , Sustancias Protectoras/metabolismo , Sustancias Protectoras/uso terapéutico , Células RAW 264.7 , Receptor de Interferón gammaRESUMEN
Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases.
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Marcación de Gen/métodos , Genoma Humano/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Reparación del Gen Blanco/métodos , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/genética , Animales , Antígenos CD34/metabolismo , ADN Complementario/genética , Endonucleasas/metabolismo , Sangre Fetal/citología , Sangre Fetal/metabolismo , Sangre Fetal/trasplante , Hematopoyesis/genética , Trasplante de Células Madre Hematopoyéticas , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Masculino , Ratones , Mutación/genética , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/terapiaRESUMEN
Mucopolysaccharidosis type I (MPS-I) is a severe genetic disease caused by a deficiency of the alpha-L-iduronidase (IDUA) enzyme. Ex vivo hematopoietic stem cell (HSC) gene therapy is a promising therapeutic approach for MPS-I, as demonstrated by preclinical studies performed in naive MPS-I mice. However, after enzyme replacement therapy (ERT), several MPS-I patients develop anti-IDUA immunity that may jeopardize ex vivo gene therapy efficacy. Here we treat MPS-I mice with an artificial immunization protocol to mimic the ERT effect in patients, and we demonstrate that IDUA-corrected HSC engraftment is impaired in pre-immunized animals by IDUA-specific CD8+ T cells spared by pre-transplant irradiation. Conversely, humoral anti-IDUA immunity does not impact on IDUA-corrected HSC engraftment. The inclusion of lympho-depleting agents in pre-transplant conditioning of pre-immunized hosts allowes rescue of IDUA-corrected HSC engraftment, which is proportional to CD8+ T cell eradication. Overall, these data demonstrate the relevance of pre-existing anti-transgene T cell immunity on ex vivo HSC gene therapy, and they suggest the application of tailored immune-depleting treatments, as well as a deeper immunological characterization of patients, to safeguard the therapeutic effects of ex vivo HSC gene therapy in immunocompetent hosts.
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Linfocitos T CD8-positivos/inmunología , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Mucopolisacaridosis I/terapia , Transgenes/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Terapia de Reemplazo Enzimático/efectos adversos , Técnicas de Inactivación de Genes , Vectores Genéticos , Humanos , Iduronidasa/genética , Iduronidasa/inmunología , Inmunidad Celular/efectos de los fármacos , Inmunización/métodos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Bazo/patologíaRESUMEN
Eradicating the malignant stem cell is the ultimate challenge in the treatment of leukaemia. Leukaemic stem cells (LSC) hijack the normal haemopoietic niche, where they are mainly protected from cytotoxic drugs. The anti-leukaemic effect of L-asparaginase (ASNase) has been extensively investigated in acute lymphoblastic leukaemia, but only partially in acute myeloid leukaemia (AML). We explored the susceptibility of AML-LSC to ASNase as well as the role of the two major cell types that constitute the bone marrow (BM) microenvironment, i.e., mesenchymal stromal cells (MSC) and monocytes/macrophages. Whilst ASNase was effective on both CD34+ CD38+ and CD34+ CD38- LSC fractions, MSC and monocytes/macrophages partially counteracted the effect of the drug. Indeed, the production of cathepsin B, a lysosomal cysteine protease, by BM monocytic cells and by AML cells classified as French-American-British M5 is related to the inactivation of ASNase. Our work demonstrates that, while MSC and monocytes/macrophages may provide a protective niche for AML cells, ASNase has a cytotoxic effect on AML blasts and, importantly, LSC subpopulations. Thus, these features should be considered in the design of future clinical studies aimed at testing ASNase efficacy in AML patients.
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Asparaginasa/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Asparaginasa/farmacología , Línea Celular , HumanosRESUMEN
Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and ß genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR "single editing" (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1157-165-specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy.
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Traslado Adoptivo , Edición Génica/métodos , Memoria Inmunológica , Mieloma Múltiple , Proteínas de Neoplasias , Fragmentos de Péptidos , Receptores de Antígenos de Linfocitos T , Linfocitos T , Animales , Línea Celular Tumoral , Femenino , Técnicas de Transferencia de Gen , Enfermedad Injerto contra Huésped , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/inmunología , Mieloma Múltiple/terapia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/trasplante , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The balance between self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells. However, the underlying molecular pathways, including the role of micro-RNA, are not completely understood. To assess the contribution of micro-RNA, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNA might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild-type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNA in the physiological stem cell-to-multi-potent progenitor transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as the most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion, as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p overexpression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiological downregulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system.
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Diferenciación Celular , Células Madre Hematopoyéticas/citología , MicroARNs/fisiología , Animales , Linaje de la Célula/genética , Análisis por Conglomerados , Cruzamientos Genéticos , Perfilación de la Expresión Génica , Hematopoyesis , Homeostasis , Humanos , Células K562 , Lentivirus/genética , Ratones , Ratones Noqueados , Estrés Oxidativo , Factor de Transcripción 1 de la Leucemia de Células Pre-B/metabolismoRESUMEN
New neurons, originating from the subventricular zone, are continuously integrating into neuronal circuitry in the olfactory bulb (OB). Using a transgenic sensor mouse, we found that adult-born OB interneurons express microRNA-125 (miR-125), whereas the pre-existing developmentally generated OB interneurons represent a unique population of cells in the adult brain, without miR-125 activity. Stable inhibition of miR-125 in newborn OB neurons resulted in enhanced dendritic morphogenesis, as well as in increased synaptic activation in response to odour sensory stimuli. These data demonstrate that miR-125 controls functional synaptic integration of adult-born OB interneurons. Our results also suggest that absence of an otherwise broadly expressed miRNA is a novel mechanism with which to achieve neuronal subtype specification.
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Células Madre Adultas/fisiología , Células Madre Embrionarias/fisiología , Interneuronas/fisiología , MicroARNs/fisiología , Bulbo Olfatorio/citología , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Diferenciación Celular/genética , Femenino , Interneuronas/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células-Madre Neurales/fisiología , Neurogénesis/genética , Bulbo Olfatorio/embriología , Bulbo Olfatorio/metabolismo , Sinapsis/genéticaRESUMEN
Chronic granulomatous disease (CGD) is a primary immunodeficiency due to a deficiency in one of the subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. CGD patients are characterized by an increased susceptibility to bacterial and fungal infections, and to granuloma formation due to the excessive inflammatory responses. Several gene therapy approaches with lentiviral vectors have been proposed but there is a lack of in vivo data on the ability to control infections and inflammation. We set up a mouse model of acute infection that closely mimic the airway infection in CGD patients. It involved an intratracheal injection of a methicillin-sensitive reference strain of S. aureus. Gene therapy, with hematopoietic stem cells transduced with regulated lentiviral vectors, restored the functional activity of NADPH oxidase complex (with 20-98% of dihydrorhodamine positive granulocytes and monocytes) and saved mice from death caused by S. aureus, significantly reducing the bacterial load and lung damage, similarly to WT mice even at low vector copy number. When challenged, gene therapy-treated XCGD mice showed correction of proinflammatory cytokines and chemokine imbalance at levels that were comparable to WT. Examined together, our results support the clinical development of gene therapy protocols using lentiviral vectors for the protection against infections and inflammation.
Asunto(s)
Terapia Genética/métodos , Enfermedad Granulomatosa Crónica/complicaciones , Trasplante de Células Madre Hematopoyéticas/métodos , Glicoproteínas de Membrana/genética , NADPH Oxidasas/genética , Neumonía Estafilocócica/terapia , Staphylococcus aureus/fisiología , Animales , Carga Bacteriana , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Vectores Genéticos/administración & dosificación , Enfermedad Granulomatosa Crónica/genética , Células Madre Hematopoyéticas/virología , Humanos , Lentivirus/genética , Ratones , NADPH Oxidasa 2 , Neumonía Estafilocócica/genética , Neumonía Estafilocócica/microbiologíaRESUMEN
Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstrated that microRNA 126 (miR-126) drives B-cell myeloid biphenotypic leukemia differentiation toward B cells without changing expression of E2A immunoglobulin enhancer-binding factor E12/E47 (E2A), early B-cell factor 1 (EBF1), or paired box protein 5, which are critical transcription factors in B-lymphopoiesis. Similar induction of B-cell differentiation by miR-126 was observed in normal hematopoietic cells in vitro and in vivo in uncommitted murine c-Kit(+)Sca1(+)Lineage(-) cells, with insulin regulatory subunit-1 acting as a target of miR-126. Importantly, in EBF1-deficient hematopoietic progenitor cells, which fail to differentiate into B cells, miR-126 significantly up-regulated B220, and induced the expression of B-cell genes, including recombination activating genes-1/2 and CD79a/b. These data suggest that miR-126 can at least partly rescue B-cell development independently of EBF1. These experiments show that miR-126 regulates myeloid vs. B-cell fate through an alternative machinery, establishing the critical role of miRNAs in the lineage specification of multipotent mammalian cells.
Asunto(s)
Linaje de la Célula/genética , Perfilación de la Expresión Génica , Leucemia de Células B/metabolismo , MicroARNs/metabolismo , Análisis de Varianza , Linfocitos B/metabolismo , Western Blotting , Trasplante de Médula Ósea , Línea Celular Tumoral , Linaje de la Célula/inmunología , Cartilla de ADN , Vectores Genéticos/genética , Humanos , Luciferasas , Células Progenitoras Mieloides , Oligonucleótidos/genética , Estadísticas no Paramétricas , Transactivadores/metabolismo , Factor de Transcripción 3/metabolismoRESUMEN
A productive immune response requires transient upregulation of the microRNA miR-155 in hematopoietic cells mediating innate and adaptive immunity. In order to investigate miR-155 in the context of tumor-associated immune responses, we stably knocked down (KD) miR-155 in the myeloid compartment of MMTV-PyMT mice, a mouse model of spontaneous breast carcinogenesis that closely mimics tumor-host interactions seen in humans. Notably, miR-155/KD significantly accelerated tumor growth by impairing classic activation of tumor-associated macrophages (TAMs). This created an imbalance toward a protumoral microenvironment as evidenced by a lower proportion of CD11c(+) TAMs, reduced expression of activation markers, and the skewing of immune cells within the tumor toward an macrophage type 2/T helper 2 response. This study highlights the importance of tumor-infiltrating hematopoietic cells in constraining carcinogenesis and establishes an antitumoral function of a prototypical oncomiR.