RESUMO
BACKGROUND: Exagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis by means of ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of BCL11A. METHODS: We conducted a phase 3, single-group, open-label study of exa-cel in patients 12 to 35 years of age with sickle cell disease who had had at least two severe vaso-occlusive crises in each of the 2 years before screening. CD34+ HSPCs were edited with the use of CRISPR-Cas9. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was freedom from severe vaso-occlusive crises for at least 12 consecutive months. A key secondary end point was freedom from inpatient hospitalization for severe vaso-occlusive crises for at least 12 consecutive months. The safety of exa-cel was also assessed. RESULTS: A total of 44 patients received exa-cel, and the median follow-up was 19.3 months (range, 0.8 to 48.1). Neutrophils and platelets engrafted in each patient. Of the 30 patients who had sufficient follow-up to be evaluated, 29 (97%; 95% confidence interval [CI], 83 to 100) were free from vaso-occlusive crises for at least 12 consecutive months, and all 30 (100%; 95% CI, 88 to 100) were free from hospitalizations for vaso-occlusive crises for at least 12 consecutive months (P<0.001 for both comparisons against the null hypothesis of a 50% response). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No cancers occurred. CONCLUSIONS: Treatment with exa-cel eliminated vaso-occlusive crises in 97% of patients with sickle cell disease for a period of 12 months or more. (CLIMB SCD-121; ClinicalTrials.gov number, NCT03745287.).
Assuntos
Anemia Falciforme , Hemoglobina Fetal , Transplante de Células-Tronco Hematopoéticas , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Adulto Jovem , Anemia Falciforme/complicações , Anemia Falciforme/genética , Anemia Falciforme/terapia , Antígenos CD34 , Bussulfano/uso terapêutico , Sistemas CRISPR-Cas , Hemoglobina Fetal/biossíntese , Hemoglobina Fetal/genética , Edição de Genes , Células-Tronco Hematopoéticas , Proteínas Repressoras , Condicionamento Pré-Transplante , Terapia Baseada em Transplante de Células e Tecidos/métodos , Agonistas Mieloablativos/uso terapêutico , Europa (Continente) , América do NorteRESUMO
FLT3, DNMT3A, and NPM1 are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific GPR56 highCD34low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (HLF). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from DNMT3A/NPM1 double-mutated subclones. Moreover, in DNMT3A R882-mutated patients, CpG hypomethylation at the HLF transcription start site correlated with high HLF mRNA expression, which was itself associated with poor survival. Loss of HLF via CRISPR/Cas9 significantly reduced the CD34+GPR56+ LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. HLF knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated HLF knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 (HES1) and the cyclin-dependent kinase inhibitor CDKN1C/p57 as novel targets of HLF, potentially mediating these effects. Overall, our data establish HLF as a novel LSC regulator in this genetically defined high-risk AML subgroup.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/genética , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Biomarcadores , Ciclo Celular/genética , Linhagem Celular Tumoral , Biologia Computacional/métodos , DNA Metiltransferase 3A , Modelos Animais de Doenças , Duplicação Gênica , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Camundongos Transgênicos , Mutação , Nucleofosmina , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sequências de Repetição em Tandem , Sítio de Iniciação de Transcrição , TranscriptomaRESUMO
Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML.
Assuntos
Herpesvirus Humano 1/imunologia , Imunidade Celular , Células Matadoras Naturais/imunologia , Leucemia/imunologia , Raios Ultravioleta , Inativação de Vírus/efeitos da radiação , Degranulação Celular/imunologia , Movimento Celular/imunologia , Feminino , Humanos , Interleucina-15/imunologia , Interleucina-2/imunologia , Células Jurkat , Masculino , NF-kappa B/imunologia , Proteína Quinase C/imunologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologiaRESUMO
Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy, which is initiated and driven by a rare fraction of leukemia stem cells (LSCs). Despite the difficulties of identifying a common LSC phenotype, there is increasing evidence that high expression of stem cell gene signatures is associated with poor clinical outcome. Identification of functionally distinct subpopulations in this disease is therefore crucial to dissecting the molecular machinery underlying LSC self-renewal. Here, we combined next-generation sequencing technology with in vivo assessment of LSC frequencies and identified the adhesion G protein-coupled receptor 56 (GPR56) as a novel and stable marker for human LSCs for the majority of AML samples. High GPR56 expression was significantly associated with high-risk genetic subgroups and poor outcome. Analysis of GPR56 in combination with CD34 expression revealed engraftment potential of GPR56(+)cells in both the CD34(-)and CD34(+)fractions, thus defining a novel LSC compartment independent of the CD34(+)CD38(-)LSC phenotype.
Assuntos
Biomarcadores Tumorais/metabolismo , Proliferação de Células , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Animais , Separação Celular , Células Cultivadas , Células HEK293 , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Células-Tronco Neoplásicas/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Análise de SobrevidaRESUMO
Retroviral integration site analysis and barcoding have been instrumental for multiplex clonal fate mapping, although their use imposes an inherent delay between sample acquisition and data analysis. Monitoring of multiple cell populations in real time would be advantageous, but multiplex assays compatible with flow cytometric tracking of competitive growth behavior are currently limited. We here describe the development and initial validation of three generations of lentiviral fluorescent genetic barcoding (FGB) systems that allow the creation of 26, 14, or 6 unique labels. Color-coded populations could be tracked in multiplex in vitro assays for up to 28 days by flow cytometry using all three vector systems. Those involving lower levels of multiplexing eased color-code generation and the reliability of vector expression and enabled functional in vitro and in vivo studies. In proof-of-principle experiments, FGB vectors facilitated in vitro multiplex screening of microRNA (miRNA)-induced growth advantages, as well as the in vivo recovery of color-coded progeny of murine and human hematopoietic stem cells. This novel series of FGB vectors provides new tools for assessing comparative growth properties in in vitro and in vivo multiplexing experiments, while simultaneously allowing for a reduction in sample numbers by up to 26-fold.
Assuntos
Rastreamento de Células/métodos , Expressão Gênica , Genes Reporter , Vetores Genéticos/genética , Lentivirus/genética , Proteínas Luminescentes/genética , Diferenciação Celular , Códon , Citometria de Fluxo , Ordem dos Genes , Técnicas de Transferência de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Proteínas Luminescentes/metabolismo , MicroRNAs/genética , Reprodutibilidade dos Testes , Transdução GenéticaRESUMO
Cord blood transplantation (CBT) recipients are at increased risk for delayed engraftment and primary graft failure, complications that are often indistinguishable early post-transplantation. Current assays fail to accurately identify recipients with slow hematopoietic recovery and distinguish them from those with pending graft failure. To address this, we prospectively examined the kinetics of immune cell subset recovery in the peripheral blood of 39 patients on days +7 and +14 after double-unit CBT (dCBT) by multiparametric flow cytometry analysis, which we term real-time immunophenotyping (RTIP). RTIP analysis at day +14 revealed distinctive patterns of reconstitution and, importantly, identified patients with slow hematopoietic recovery who went on to engraft. Strikingly, higher absolute numbers of circulating monocytes and natural killer cells at day +14 were predictive of engraftment, but only the absolute number of circulating monocytes was significantly correlated with time to engraftment. This is the first evidence that RTIP on patient peripheral blood mononuclear cells early after dCBT is technically feasible and can be used as a "signature" for predicting the kinetics of hematopoietic recovery. Furthermore, RTIP is a time- and cost-efficient methodology that has the potential to become a clinically feasible diagnostic tool to guide therapeutic interventions in high-risk patients; therefore, its utility should be evaluated in a large cohort of patients.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Sobrevivência de Enxerto , Imunofenotipagem , Leucócitos/citologia , Adolescente , Adulto , Criança , Transplante de Células-Tronco de Sangue do Cordão Umbilical/normas , Função Retardada do Enxerto , Feminino , Citometria de Fluxo , Humanos , Cinética , Leucócitos/imunologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Adulto JovemRESUMO
Leukemic transformation of human cells is a complex process. Here we show that forced expression of MN1 in primitive human cord blood cells maintained on stromal cells in vitro induces a transient, but not serially transplantable, myeloproliferation in engrafted mice. However, cotransduction of an activated HOX gene (NUP98HOXD13) with MN1 induces a serially transplantable acute myeloid leukemia (AML). Further characterization of the leukemic cells generated from the dually transduced cells showed the activation of stem cell gene expression signatures also found in primary human AML. These findings show a new forward genetic model of human leukemogenesis and further highlight the relevance of homeobox transcription factors in the transformation process.
Assuntos
Transformação Celular Neoplásica/metabolismo , Sangue Fetal/metabolismo , Leucemia Mieloide Aguda/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas de Fusão Oncogênica/genética , Transativadores , Proteínas Supressoras de Tumor/genéticaRESUMO
Pre-B-cell leukemia homeobox interacting protein 1 or human PBX1 interacting protein (PBXIP1/HPIP) is a co-repressor of pre-B-cell leukemia homeobox 1 (PBX1) and is also known to regulate estrogen receptor functions by associating with the microtubule network. Despite its initial discovery in the context of hematopoietic cells, little is yet known about the role of HPIP in hematopoiesis. Here, we show that lentivirus-mediated overexpression of HPIP in human CD34(+) cells enhances hematopoietic colony formation in vitro, whereas HPIP knockdown leads to a reduction in the number of such colonies. Interestingly, erythroid colony number was significantly higher in HPIP-overexpressing cells. In addition, forced expression of HPIP in K562 cells, a multipotent erythro-megakaryoblastic leukemia cell line, led to an induction of erythroid differentiation. HPIP overexpression in both CD34(+) and K562 cells was associated with increased activation of the PI3K/AKT pathway, and corresponding treatment with a PI3K-specific inhibitor, LY-294002, caused a reduction in clonogenic progenitor number in HPIP-expressing CD34(+) cells and decreased K562 cell differentiation. Combined, these findings point to an important role of the PI3K/AKT pathway in mediating HPIP-induced effects on the growth and differentiation of hematopoietic cells. Interestingly, HPIP gene expression was found to be induced in K562 cells in response to erythroid differentiation signals such as DMSO and erythropoietin. The erythroid lineage-specific transcription factor GATA1 binds to the HPIP promoter and activates HPIP gene transcription in a CCCTC-binding factor (CTCF)-dependent manner. Co-immunoprecipitation and co-localization experiments revealed the association of CTCF with GATA1 indicating the recruitment of CTCF/GATA1 transcription factor complex onto the HPIP promoter. Together, this study provides evidence that HPIP is a target of GATA1 and CTCF in erythroid cells and plays an important role in erythroid differentiation by modulating the PI3K/AKT pathway.
Assuntos
Diferenciação Celular , Células Eritroides/citologia , Células Eritroides/metabolismo , Fatores de Transcrição/metabolismo , Antígenos CD34/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Fator de Ligação a CCCTC , Diferenciação Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Proteínas Correpressoras , Dimetil Sulfóxido/farmacologia , Células Eritroides/efeitos dos fármacos , Eritropoetina/farmacologia , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HL-60 , Hematopoese/efeitos dos fármacos , Humanos , Células K562 , Células Mieloides/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.
Assuntos
Receptor 1 de Folato , Imunoterapia Adotiva , Leucemia Megacarioblástica Aguda , Proteínas de Fusão Oncogênica , Animais , Criança , Pré-Escolar , Humanos , Lactente , Modelos Animais de Doenças , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Leucemia Megacarioblástica Aguda/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Linfócitos T , Transcriptoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.
Assuntos
Antineoplásicos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azacitidina/farmacologia , Evolução Clonal , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/normas , Sinergismo Farmacológico , Feminino , GTP Fosfo-Hidrolases/genética , Humanos , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/mortalidade , Leucemia Mielomonocítica Crônica/patologia , Proteínas de Membrana/genética , Camundongos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridonas/farmacologia , Piridonas/uso terapêutico , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , RNA Interferente Pequeno/genética , Receptor Notch1/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
PURPOSE: A cryptic inv(16)(p13.3q24.3) encoding the CBFA2T3-GLIS2 fusion is associated with poor outcome in infants with acute megakaryocytic leukemia. We aimed to broaden our understanding of the pathogenesis of this fusion through transcriptome profiling. EXPERIMENTAL DESIGN: Available RNA from children and young adults with de novo acute myeloid leukemia (AML; N = 1,049) underwent transcriptome sequencing (mRNA and miRNA). Transcriptome profiles for those with the CBFA2T3-GLIS2 fusion (N = 24) and without (N = 1,025) were contrasted to define fusion-specific miRNAs, genes, and pathways. Clinical annotations defined distinct fusion-associated disease characteristics and outcomes. RESULTS: The CBFA2T3-GLIS2 fusion was restricted to infants <3 years old (P < 0.001), and the presence of this fusion was highly associated with adverse outcome (P < 0.001) across all morphologic classifications. Further, there was a striking paucity of recurrent cooperating mutations, and transduction of cord blood stem cells with this fusion was sufficient for malignant transformation. CBFA2T3-GLIS2 positive cases displayed marked upregulation of genes with cell membrane/extracellular matrix localization potential, including NCAM1 and GABRE. Additionally, miRNA profiling revealed significant overexpression of mature miR-224 and miR-452, which are intronic miRNAs transcribed from the GABRE locus. Gene-set enrichment identified dysregulated Hippo, TGFß, and hedgehog signaling, as well as NCAM1 (CD56) interaction pathways. Therapeutic targeting of fusion-positive leukemic cells with CD56-directed antibody-drug conjugate caused significant cytotoxicity in leukemic blasts. CONCLUSIONS: The CBFA2T3-GLIS2 fusion defines a highly refractory entity limited to infants that appears to be sufficient for malignant transformation. Transcriptome profiling elucidated several highly targetable genes and pathways, including the identification of CD56, providing a highly plausible target for therapeutic intervention.
Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , Mutação , Proteínas de Fusão Oncogênica/genética , Adulto , Antígeno CD56/genética , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro , Receptores de GABA-A/genética , Adulto JovemRESUMO
The ability to expand hematopoietic stem and progenitor cells (HSPCs) ex vivo is critical to fully realize the potential of HSPC-based therapies. In particular, the application of clinically effective therapies, such as cord blood transplantation, has been impeded because of limited HSPC availability. Here, using 3D culture of human HSPCs in a degradable zwitterionic hydrogel, we achieved substantial expansion of phenotypically primitive CD34+ cord blood and bone-marrow-derived HSPCs. This culture system led to a 73-fold increase in long-term hematopoietic stem cell (LT-HSC) frequency, as demonstrated by limiting dilution assays, and the expanded HSPCs were capable of hematopoietic reconstitution for at least 24 weeks in immunocompromised mice. Both the zwitterionic characteristics of the hydrogel and the 3D format were important for HSPC self-renewal. Mechanistically, the impact of 3D zwitterionic hydrogel culture on mitigating HSPC differentiation and promoting self-renewal might result from an inhibition of excessive reactive oxygen species (ROS) production via suppression of O2-related metabolism. HSPC expansion using zwitterionic hydrogels has the potential to facilitate the clinical application of hematopoietic-stem-cell therapies.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Hematopoéticas/citologia , Hidrogéis/farmacologia , Animais , Antígenos CD34/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Espécies Reativas de Oxigênio/metabolismoAssuntos
Leucemia Megacarioblástica Aguda , Humanos , Leucemia Megacarioblástica Aguda/imunologia , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/patologia , Camundongos , Animais , Criança , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Imunoterapia Adotiva , Feminino , Masculino , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/imunologia , Proteínas de Fusão Oncogênica/metabolismoRESUMO
Enhanced gene transfer efficiencies and higher yields of transplantable transduced human hematopoietic stem cells are continuing goals for improving clinical protocols that use stemcell-based gene therapies. Here, we examined the effect of the HSC agonist UM171 on these endpoints in both in vitro and in vivo systems. Using a 22-hr transduction protocol, we found that UM171 significantly enhances both the lentivirus-mediated transduction and yield of CD34+ and CD34+CD45RA- hematopoietic cells from human cord blood to give a 6-fold overall higher recovery of transduced hematopoietic stem cells, including cells with long-term lympho-myeloid repopulating activity in immunodeficient mice. The ability of UM171 to enhance gene transfer to primitive cord blood hematopoietic cells extended to multiple lentiviral pseudotypes, gamma retroviruses, and non-integrating lentiviruses and to adult bone marrow cells. UM171, thus, provides an interesting reagent for improving the ex vivo production of gene-modified cells and for reducing requirements of virus for a broad range of applications.
RESUMO
Transplantation of genetically corrected autologous hematopoietic stem cells is an attractive approach for the cure of sickle-cell disease and beta-thalassemia. Here, we infected human cord blood cells with a self-inactivating lentiviral vector encoding an anti-sickling betaA-T87Q-globin transgene and analyzed the transduced progeny produced over a 6-month period after transplantation of the infected cells directly into sublethally irradiated NOD/LtSz-scid/scid mice. Approximately half of the human erythroid and myeloid progenitors regenerated in the mice containing the transgene, and erythroid cells derived in vitro from these in vivo-regenerated cells produced high levels of betaA-T87Q-globin protein. Linker-mediated PCR analysis identified multiple transgene-positive clones in all mice analyzed with 2.1 +/- 0.1 integrated proviral copies per cell. Genomic sequencing of vector-containing fragments showed that 86% of the proviral inserts had occurred within genes, including several genes implicated in human leukemia. These findings indicate effective transduction of very primitive human cord blood cells with a candidate therapeutic lentiviral vector resulting in the long-term and robust, erythroid-specific production of therapeutically relevant levels of beta-globin protein. However, the frequency of proviral integration within genes that regulate hematopoiesis points to a need for additional safety modifications.
Assuntos
Sangue Fetal/citologia , Vetores Genéticos/metabolismo , Globinas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Lentivirus/metabolismo , Transdução Genética , Animais , Linhagem da Célula , Transplante de Células , Células Cultivadas , Cromossomos Humanos , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Globinas/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Lentivirus/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , TransgenesRESUMO
BACKGROUND AND OBJECTIVES: Primitive human hematopoietic cells contain higher levels of aldehyde dehydrogenase (ALDH) activity than their terminally differentiating progeny but the particular stages when ALDH levels change have not been well defined. The objective of this study was to compare ALDH levels among the earliest stages of hematopoietic cell differentiation and to determine whether these could be exploited to obtain improved purity of human cord blood cells with long-term lympho-myeloid repopulating activity in vivo. DESIGN AND METHODS: ALDEFLUOR-stained human cord blood cells displaying different levels of ALDH activity were first analyzed for co-expression of various surface markers. Subsets of these cells were then isolated by multi-parameter flow cytometry and assessed for short-and long-term repopulating activity in sublethally irradiated immunodeficient mice. RESULTS: Most short-term myeloid repopulating cells (STRC-M) and all long-term lympho-myeloid repopulating cells (LTRC-ML) stained selectively as ALDH+. Limiting dilution analysis of the frequencies of both STRC-M and LTRC-ML showed that they were similarly and most highly enriched in the 10% top ALDH+ cells. Removal of cells expressing CD2, CD3, CD7, CD14, CD16, CD24, CD36, CD38, CD56, CD66b, or glycophorin A from the ALDH+ low-density fraction of human cord blood cells with low light side-scattering properties yielded a population containing LTRC-ML at a frequency of 1/360. INTERPRETATION AND CONCLUSION: Elevated ALDH activity is a broadly inclusive property of primitive human cord blood cells that, in combination with other markers, allows easy isolation of the stem cell fraction at unprecedented purities.
Assuntos
Aldeído Desidrogenase/metabolismo , Separação Celular/métodos , Células-Tronco Hematopoéticas/enzimologia , Animais , Células da Medula Óssea/enzimologia , Sangue Fetal/enzimologia , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
The hematopoietic syndrome of acute radiation syndrome (h-ARS) is characterized by severe bone marrow aplasia, resulting in a significant risk for bleeding, infections, and death. To date, clinical management of h-ARS is limited to supportive care dictated by the level of radiation exposure, with a high incidence of mortality in those exposed to high radiation doses. The ideal therapeutic agent would be an immediately available, easily distributable single-agent therapy capable of rapid in vivo hematopoietic reconstitution until recovery of autologous hematopoiesis occurs. Using a murine model of h-ARS, we herein demonstrate that infusion of ex vivo expanded murine hematopoietic stem and progenitor cells (HSPCs) into major histocompatibility complex mismatched recipient mice exposed to a lethal dose of ionizing radiation (IR) led to rapid myeloid recovery and improved survival. Survival benefit was significant in a dose-dependent manner even when infusion of the expanded cell therapy was delayed 3 days after lethal IR exposure. Most surviving mice (80%) demonstrated long-term in vivo persistence of donor T cells at low levels, and none had evidence of graft versus host disease. Furthermore, survival of donor-derived skin grafts was significantly prolonged in recipients rescued from h-ARS by infusion of the mismatched expanded cell product. These findings provide evidence that ex vivo expanded mismatched HSPCs can provide rapid, high-level hematopoietic reconstitution, mitigate IR-induced mortality, and convey donor-specific immune tolerance in a murine h-ARS model. Stem Cells Translational Medicine 2017;6:566-575.
Assuntos
Síndrome Aguda da Radiação/terapia , Proliferação de Células , Sobrevivência de Enxerto , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos da radiação , Antígenos de Histocompatibilidade/imunologia , Histocompatibilidade , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Transplante de Pele , Tolerância ao Transplante , Síndrome Aguda da Radiação/imunologia , Síndrome Aguda da Radiação/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Recuperação de Função Fisiológica , Transdução de Sinais , Fatores de TempoRESUMO
The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.
Assuntos
Sangue Fetal/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Indóis/farmacologia , Pirimidinas/farmacologia , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Regeneração/efeitos dos fármacos , Animais , Técnicas de Cultura de Células , Sangue Fetal/citologia , Sangue Fetal/fisiologia , Terapia Genética/métodos , Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/fisiologia , Humanos , Hospedeiro Imunocomprometido , Indóis/química , Camundongos , Pirimidinas/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Aldehyde dehydrogenase (ALDH) activity is a widely used marker for human hematopoietic stem cells (HSCs), yet its relevance and role in murine HSCs remain unclear. We found that murine marrow cells with a high level of ALDH activity as measured by Aldefluor staining (ALDH(br) cells) do not contain known HSCs or progenitors. In contrast, highly enriched murine HSCs defined by the CD48(-)EPCR(+) and other phenotypes contain two subpopulations, one that stains dimly with Aldefluor (ALDH(dim)) and one that stains at intermediate levels (ALDH(int)). The CD48(-)EPCR(+)ALDH(dim) cells are virtually all in G(0) and yield high levels of engraftment via both intravenous and intrabone routes. In contrast the CD48(-)EPCR(+)ALDH(int) cells are virtually all in G(1), have little intravenous engraftment potential, and yet can engraft long-term after intrabone transplantation. These data demonstrate that Aldefluor staining of unfractionated murine marrow does not identify known HSCs or progenitors. However, varying levels of Aldefluor staining when combined with CD48 and EPCR detection can identify novel populations in murine marrow including a highly enriched population of resting HSCs and a previously unknown HSC population in G(1) with an intravenous engraftment defect.