RESUMEN
Transcriptome sequencing has identified multiple subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL) of prognostic significance, but a minority of cases lack a known genetic driver. Here, we used integrated whole-genome (WGS) and -transcriptome sequencing (RNA-seq), enhancer mapping, and chromatin topology analysis to identify previously unrecognized genomic drivers in B-ALL. Newly diagnosed (n = 3221) and relapsed (n = 177) B-ALL cases with tumor RNA-seq were studied. WGS was performed to detect mutations, structural variants, and copy number alterations. Integrated analysis of histone 3 lysine 27 acetylation and chromatin looping was performed using HiChIP. We identified a subset of 17 newly diagnosed and 5 relapsed B-ALL cases with a distinct gene expression profile and 2 universal and unique genomic alterations resulting from aberrant recombination-activating gene activation: a focal deletion downstream of PAN3 at 13q12.2 resulting in CDX2 deregulation by the PAN3 enhancer and a focal deletion of exons 18-21 of UBTF at 17q21.31 resulting in a chimeric fusion, UBTF::ATXN7L3. A subset of cases also had rearrangement and increased expression of the PAX5 gene, which is otherwise uncommon in B-ALL. Patients were more commonly female and young adult with median age 35 (range,12-70 years). The immunophenotype was characterized by CD10 negativity and immunoglobulin M positivity. Among 16 patients with known clinical response, 9 (56.3%) had high-risk features including relapse (n = 4) or minimal residual disease >1% at the end of remission induction (n = 5). CDX2-deregulated, UBTF::ATXN7L3 rearranged (CDX2/UBTF) B-ALL is a high-risk subtype of leukemia in young adults for which novel therapeutic approaches are required.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adolescente , Adulto , Anciano , Factor de Transcripción CDX2/genética , Niño , Cromatina , Femenino , Genómica/métodos , Humanos , Masculino , Persona de Mediana Edad , Proteínas del Complejo de Iniciación de Transcripción Pol1 , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pronóstico , Factores de Transcripción/genética , Transcriptoma , Adulto JovenRESUMEN
Infection is a major cause of treatment-related morbidity and mortality in pediatric acute lymphoblastic leukemia (ALL). Most children with ALL who develop life-threatening bacterial infections do so during induction therapy. We describe a rare case of ALL presenting simultaneously with Streptococcus agalactiae group B Streptococcus bacteremia and meningitis in a 3-year-old girl. She received appropriate antimicrobial therapy and a 2-drug early induction regimen consisting of vincristine and dexamethasone, leading to slow neurologic recovery and a favorable initial response to anti-neoplastic therapy as evidenced by minimal residual disease of 1.12% on day 15 of induction.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Dexametasona , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Infecciones Estreptocócicas , Streptococcus agalactiae , Humanos , Femenino , Preescolar , Infecciones Estreptocócicas/tratamiento farmacológico , Infecciones Estreptocócicas/microbiología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/complicaciones , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Dexametasona/uso terapéutico , Dexametasona/administración & dosificación , Meningitis Bacterianas/tratamiento farmacológico , Meningitis Bacterianas/microbiología , Meningitis Bacterianas/diagnóstico , Vincristina/administración & dosificación , Vincristina/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicaciones , Bacteriemia/tratamiento farmacológico , Bacteriemia/microbiologíaRESUMEN
BACKGROUND: Outcomes for children with relapsed or refractory acute myeloid leukaemia remain poor. The BCL-2 inhibitor, venetoclax, has shown promising activity in combination with hypomethylating agents and low-dose cytarabine in older adults for whom chemotherapy is not suitable with newly diagnosed acute myeloid leukaemia. We aimed to determine the safety and explore the activity of venetoclax in combination with standard and high-dose chemotherapy in paediatric patients with relapsed or refractory acute myeloid leukaemia. METHODS: We did a phase 1, dose-escalation study at three research hospitals in the USA. Eligible patients were aged 2-22 years with relapsed or refractory acute myeloid leukaemia or acute leukaemia of ambiguous lineage with adequate organ function and performance status. During dose escalation, participants received venetoclax orally once per day in continuous 28-day cycles at either 240 mg/m2 or 360 mg/m2, in combination with cytarabine received intravenously every 12 h at either 100 mg/m2 for 20 doses or 1000 mg/m2 for eight doses, with or without intravenous idarubicin (12 mg/m2) as a single dose, using a rolling-6 accrual strategy. The primary endpoint was the recommended phase 2 dose of venetoclax plus chemotherapy and the secondary endpoint was the proportion of patients treated at the recommended phase 2 dose who achieved complete remission or complete remission with incomplete haematological recovery. Analyses were done on patients who received combination therapy. The study is registered with ClinicalTrials.gov (NCT03194932) and is now enrolling to address secondary and exploratory objectives. FINDINGS: Between July 1, 2017, and July 2, 2019, 38 patients were enrolled (aged 3-22 years; median 10 [IQR 7-13]), 36 of whom received combination therapy with dose escalation, with a median follow-up of 7·1 months (IQR 5·1-11·2). The recommended phase 2 dose of venetoclax was found to be 360 mg/m2 (maximum 600 mg) combined with cytarabine (1000 mg/m2 per dose for eight doses), with or without idarubicin (12 mg/m2 as a single dose). Overall responses were observed in 24 (69%) of the 35 patients who were evaluable after cycle 1. Among the 20 patients treated at the recommended phase 2 dose, 14 (70%, 95% CI 46-88) showed complete response with or without complete haematological recovery, and two (10%) showed partial response. The most common grade 3-4 adverse events were febrile neutropenia (22 [66%]), bloodstream infections (six [16%]), and invasive fungal infections (six [16%]). Treatment-related death occurred in one patient due to colitis and sepsis. INTERPRETATION: The safety and activity of venetoclax plus chemotherapy in paediatric patients with heavily relapsed and refractory acute myeloid leukaemia suggests that this combination should be tested in newly diagnosed paediatric patients with high-risk acute myeloid leukaemia. FUNDING: US National Institutes of Health, American Lebanese Syrian Associated Charities, AbbVie, and Gateway for Cancer Research.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Citarabina/administración & dosificación , Idarrubicina/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Sulfonamidas/administración & dosificación , Adolescente , Niño , Preescolar , Femenino , Humanos , Masculino , Adulto JovenRESUMEN
BACKGROUND: Novel therapies are urgently needed for pediatric patients with relapsed acute myeloid leukemia (AML). METHODS: To determine whether the histone deacetylase inhibitor panobinostat could be safely given in combination with intensive chemotherapy, a phase 1 trial was performed in which 17 pediatric patients with relapsed or refractory AML received panobinostat (10, 15, or 20 mg/m2 ) before and in combination with fludarabine and cytarabine. RESULTS: All dose levels were tolerated, with no dose-limiting toxicities observed at any dose level. Pharmacokinetic studies demonstrated that exposure to panobinostat was proportional to the dose given, with no associations between pharmacokinetic parameters and age, weight, or body surface area. Among the 9 patients who had sufficient (>2%) circulating blasts on which histone acetylation studies could be performed, 7 demonstrated at least 1.5-fold increases in acetylation. Although no patients had a decrease in circulating blasts after single-agent panobinostat, 8 of the 17 patients (47%), including 5 of the 6 patients treated at dose level 3, achieved complete remission. Among the 8 complete responders, 6 (75%) attained negative minimal residual disease status. CONCLUSIONS: Panobinostat can be safely administered with chemotherapy and results in increased blast histone acetylation. This suggests that it should be further studied in AML.
Asunto(s)
Panobinostat/farmacología , Panobinostat/farmacocinética , Panobinostat/uso terapéutico , Adolescente , Adulto , Niño , Femenino , Humanos , Leucemia Mieloide Aguda , Masculino , Recurrencia Local de Neoplasia , Adulto JovenRESUMEN
Studies in diverse organisms have revealed a surprising depth to the evolutionary conservation of genetic modules. For example, a systematic analysis of such conserved modules has recently shown that genes in yeast that maintain cell walls have been repurposed in vertebrates to regulate vein and artery growth. We reasoned that by analyzing this particular module, we might identify small molecules targeting the yeast pathway that also act as angiogenesis inhibitors suitable for chemotherapy. This insight led to the finding that thiabendazole, an orally available antifungal drug in clinical use for 40 years, also potently inhibits angiogenesis in animal models and in human cells. Moreover, in vivo time-lapse imaging revealed that thiabendazole reversibly disassembles newly established blood vessels, marking it as vascular disrupting agent (VDA) and thus as a potential complementary therapeutic for use in combination with current anti-angiogenic therapies. Importantly, we also show that thiabendazole slows tumor growth and decreases vascular density in preclinical fibrosarcoma xenografts. Thus, an exploration of the evolutionary repurposing of gene networks has led directly to the identification of a potential new therapeutic application for an inexpensive drug that is already approved for clinical use in humans.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antifúngicos/farmacología , Redes Reguladoras de Genes , Tiabendazol/farmacología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Fibrosarcoma/metabolismo , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Inmunohistoquímica , Lovastatina/farmacología , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Imagen de Lapso de Tiempo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Xenopus laevis/genética , Xenopus laevis/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Minimal residual disease detection provides critical prognostic predictor of treatment outcome and is the standard of care for B lymphoblastic leukemia. Flow cytometry-based minimal residual disease detection is the most common test modality and has high sensitivity (0.01%) and a rapid turnaround time (24 hours). This article details the leukemia associated immunophenotype analysis approach for flow cytometry-based minimal residual disease detection used at St. Jude Children's Research Hospital and importance of using guide gates and back-gating.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras B , Niño , Humanos , Neoplasia Residual/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Resultado del Tratamiento , Pronóstico , Citometría de FlujoRESUMEN
Transposon-mediated integration strategies in Xenopus offer simple and robust methods for the generation of germline transgenic animals. Co-injection of fertilized one-cell embryos with plasmid DNA harboring a transposon transgene and synthetic mRNA encoding the cognate transposase enzyme results in mosaic integration of the transposon at early cleavage stages that are frequently passed through the germline in the adult animal. Micro-injection of fertilized embryos is a routine procedure used by many laboratories that use Xenopus as a developmental model and, as such, the transposon transgenesis method can be performed without additional equipment or specialized methodologies. The methods for injecting Xenopus embryos are well documented in the literature so here we provide a step-by-step guide to other aspects of transposon transgenesis, including screening mosaic founders for germline transmission of the transgene and general husbandry considerations related to management of populations of transgenic frogs.
Asunto(s)
Elementos Transponibles de ADN , Xenopus/metabolismo , Animales , Animales Modificados Genéticamente , Bacteriófagos/metabolismo , Cruzamientos Genéticos , ADN/metabolismo , Silenciador del Gen , Oocitos/metabolismo , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Transgenes , Transposasas/metabolismoRESUMEN
Minimal residual disease detection provides critical prognostic predictor of treatment outcome and is the standard of care for B lymphoblastic leukemia. Flow cytometry-based minimal residual disease detection is the most common test modality and has high sensitivity (0.01%) and a rapid turnaround time (24 hours). This article details the leukemia associated immunophenotype analysis approach for flow cytometry-based minimal residual disease detection used at St. Jude Children's Research Hospital and importance of using guide gates and back-gating.
Asunto(s)
Laboratorios , Leucemia-Linfoma Linfoblástico de Células Precursoras , Niño , Citometría de Flujo , Humanos , Inmunofenotipificación , Neoplasia ResidualRESUMEN
Lineage-ambiguous leukemias are high-risk malignancies of poorly understood genetic basis. Here, we describe a distinct subgroup of acute leukemia with expression of myeloid, T lymphoid, and stem cell markers driven by aberrant allele-specific deregulation of BCL11B, a master transcription factor responsible for thymic T-lineage commitment and specification. Mechanistically, this deregulation was driven by chromosomal rearrangements that juxtapose BCL11B to superenhancers active in hematopoietic progenitors, or focal amplifications that generate a superenhancer from a noncoding element distal to BCL11B. Chromatin conformation analyses demonstrated long-range interactions of rearranged enhancers with the expressed BCL11B allele and association of BCL11B with activated hematopoietic progenitor cell cis-regulatory elements, suggesting BCL11B is aberrantly co-opted into a gene regulatory network that drives transformation by maintaining a progenitor state. These data support a role for ectopic BCL11B expression in primitive hematopoietic cells mediated by enhancer hijacking as an oncogenic driver of human lineage-ambiguous leukemia. SIGNIFICANCE: Lineage-ambiguous leukemias pose significant diagnostic and therapeutic challenges due to a poorly understood molecular and cellular basis. We identify oncogenic deregulation of BCL11B driven by diverse structural alterations, including de novo superenhancer generation, as the driving feature of a subset of lineage-ambiguous leukemias that transcend current diagnostic boundaries.This article is highlighted in the In This Issue feature, p. 2659.
Asunto(s)
Elementos de Facilitación Genéticos , Leucemia Mieloide Aguda , Proteínas Represoras , Proteínas Supresoras de Tumor , Redes Reguladoras de Genes , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND: The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency. RESULTS: To test whether Tol2 transposons integrated in the Xenopus tropicalis genome are substrates for remobilization, we injected in vitro transcribed Tol2 mRNA into one-cell embryos harbouring a single copy of a Tol2 transposon. Integration site analysis of injected embryos from two founder lines showed at least one somatic remobilization event per embryo. We also demonstrate that the remobilized transposons are transmitted through the germline and re-integration can result in the generation of novel GFP expression patterns in the developing tadpole. Although the parental line contained a single Tol2 transposon, the resulting remobilized tadpoles frequently inherit multiple copies of the transposon. This is likely to be due to the Tol2 transposase acting in discrete blastomeres of the developing injected embryo during the cell cycle after DNA synthesis but prior to mitosis. CONCLUSIONS: In this study, we demonstrate that single copy Tol2 transposons integrated into the Xenopus tropicalis genome are effective substrates for excision and random re-integration and that the remobilized transposons are transmitted through the germline. This is an important step in the development of 'transposon hopping' strategies for insertional mutagenesis, gene trap and enhancer trap screens in this highly tractable developmental model organism.
Asunto(s)
Elementos Transponibles de ADN , Mutagénesis Insercional/métodos , Xenopus/genética , Animales , Embrión no Mamífero/metabolismo , Mutación de Línea Germinal , Modelos Animales , Xenopus/embriologíaRESUMEN
The cell membrane glycolipid GD2 is expressed by multiple solid tumors, including 88% of osteosarcomas and 98% of neuroblastomas. However, osteosarcomas are highly heterogeneous, with many tumors exhibiting GD2 expression on <50% of the individual cells, while some tumors are essentially GD2-negative. Anti-GD2 immunotherapy is the current standard of care for high-risk neuroblastoma, but its application to recurrent osteosarcomas, for which no effective therapies exist, has been extremely limited. This is, in part, because the standard assays to measure GD2 expression in these heterogeneous tumors are not quantitative and are subject to tissue availability and sampling bias. To address these limitations, we evaluated a novel, sensitive radiotracer [64Cu]Cu-Bn-NOTA-hu14.18K322A to detect GD2 expression in osteosarcomas (six patient-derived xenografts and one cell line) in vivo using positron emission tomography (PET). Tumor uptake of the radiolabeled, humanized anti-GD2 antibody [64Cu]Cu-Bn-NOTA-hu14.18K322A was 7-fold higher in modestly GD2-expressing osteosarcomas (32% GD2-positive cells) than in a GD2-negative tumor (9.8% vs. 1.3% of the injected dose per cc, respectively). This radiotracer also identified lesions as small as 29 mm3 in a 34% GD2-positive model of metastatic osteosarcoma of the lung. Radiolabeled antibody accumulation in patient-derived xenografts correlated with GD2 expression as measured by flow cytometry (Pearson r = 0.88, P = 0.01), distinguishing moderately GD2-expressing osteosarcomas (32%-69% GD2-positive cells) from high GD2 expressors (>99%, P < 0.05). These results support the utility of GD2 imaging with PET to measure GD2 expression in osteosarcoma and thus maximize the clinical impact of anti-GD2 immunotherapy. SIGNIFICANCE: In situ assessment of all GD2-positive osteosarcoma sites with a novel PET radiotracer could significantly impact anti-GD2 immunotherapy patient selection and enable noninvasive probing of correlations between target expression and therapeutic response.
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Anticuerpos Monoclonales/farmacología , Neoplasias Óseas/patología , Gangliósidos/antagonistas & inhibidores , Neoplasias Pulmonares/secundario , Recurrencia Local de Neoplasia/patología , Osteosarcoma/patología , Tomografía de Emisión de Positrones/métodos , Animales , Apoptosis , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/inmunología , Neoplasias Óseas/metabolismo , Proliferación Celular , Gangliósidos/inmunología , Humanos , Inmunoterapia , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/metabolismo , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/inmunología , Osteosarcoma/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Mix/Bix family of Pax-like homeodomain transcription factors is expressed early in vertebrate development and play important roles in endoderm and mesoderm formation. Like other Pax-related homeodomain proteins, the Mix/Bix family binds DNA as monomers or dimers and dimerization is mediated by the homeodomain. While the Mix/Bix family shares extensive sequence homology within the DNA-binding homeodomain, ectopic expression of these proteins has profoundly different outcomes. Expression of Xenopus Mix.3/Mixer in explanted ectoderm results in endoderm differentiation, whereas Mix.1 expression does not. In this study we sought to define the domains of Mix.3/Mixer that are responsible for this endoderm inducing activity. We generated domain swap mutants between Mix.3/Mixer and Mix.1 and tested their ability to induce endoderm in explanted ectoderm. We demonstrate that the homeodomain and sixty-two amino acids in the carboxyl terminus are required to induce endoderm and that these domains must be on the same polypeptide and can not act in trans as a heterodimer. A Smad2 interaction motif in Mix.3/Mixer is involved in endoderm differentiation but is not essential. Thus, we have defined the regions of Mix.3/Mixer that confer endoderm-inducing activity. These studies reveal a novel co-operation between the homeodomain and a small domain in the carboxyl terminal region that is essential for Mix.3/Mixer function.
Asunto(s)
Endodermo/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Animales , Secuencia de Bases , ADN/genética , ADN/metabolismo , Inducción Embrionaria , Proteínas Fetales/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Mutagénesis , Unión Proteica , Estructura Terciaria de Proteína , Proteína Smad2/metabolismo , Proteínas de Dominio T Box/genética , Factores de Transcripción/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Cytokine signaling plays an important role in the survival and differentiation of vertebrate hematopoietic cells. In red blood cells, erythropoietin is a key component of the differentiation program and maintains the homeostasis of the erythroid compartment. In the adult, anemia stimulates high levels of circulating erythropoietin that drives erythropoiesis to restore normal levels of red blood cells in circulation. Erythropoietin activates the erythropoietin receptor on immature red blood cell precursors to promote their survival and differentiation. Although extensively studied in mammalian systems, a complete understanding of the function of the erythropoietin receptor during primitive erythropoiesis has been lacking. To address this problem, we have cloned the Xenopus laevis erythropoietin receptor in order to further understand the development of primitive erythropoiesis. The amphibian erythropoietin receptor shares 33% amino acid sequence identity with the mammalian erythropoietin receptors and contains the conserved extracellular ligand binding and fibronectin domains, the WSXWS motif common to cytokine receptors, and several tyrosine phosphorylation sites located on the intracellular domain of the receptor. Expression of the erythropoietin receptor is first detected by in situ hybridization in the ventral blood island during tailbud stages.
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Clonación Molecular , Regulación del Desarrollo de la Expresión Génica , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Xenopus/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Northern Blotting , Codón Iniciador , Cisteína/química , Bases de Datos Factuales , Embrión no Mamífero , Exones , Etiquetas de Secuencia Expresada , Hibridación in Situ , Intrones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN Mensajero , Receptores de Eritropoyetina/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Tirosina/química , Xenopus/embriología , Xenopus/crecimiento & desarrolloRESUMEN
The Evi-1 gene was first identified as a site for viral integration in murine myeloid leukemia. Evi-1 is a zinc finger transcription factor that has been implicated in the development of myeloid neoplasia. In humans, disruption of the Evi-1 locus, by chromosomal rearrangements, is associated with myeloid leukemia and myelodyplastic syndromes. Here, we report the cloning and developmental pattern of expression of Xenopus Evi-1. xEvi-1 is expressed during oogenesis and during embryonic development. In situ hydridization reveals that xEvi-1 has a dynamic expression profile during early embryonic development. Expression of Evi-1 is detected by in situ hybridization in the pronephric tissue, the brain and in neural crest derivatives of the head and neck.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/biosíntesis , Secuencia de Aminoácidos , Animales , Northern Blotting , Encéfalo/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Biblioteca de Genes , Hibridación in Situ , Riñón/embriología , Proteína del Locus del Complejo MDS1 y EV11 , Ratones , Datos de Secuencia Molecular , Cresta Neural/metabolismo , Oocitos/metabolismo , Poli A/química , Proto-Oncogenes , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transcripción Genética , Xenopus laevisRESUMEN
Patients with severe sickle cell disease (SCD) are candidates for gene therapy using autologous hematopoietic stem cells (HSCs), but concomitant multi-organ disease may contraindicate pretransplant conditioning with full myeloablation. We tested whether nonmyeloablative conditioning, a regimen used successfully for allogeneic bone marrow transplantation of adult SCD patients, allows engraftment of γ-globin gene-corrected cells to a therapeutic level in the Berkeley mouse model of SCD. Animals transplanted according to this regimen averaged 35% engraftment of transduced hematopoietic stem cells with an average vector copy < 2.0. Fetal hemoglobin (HbF) levels ranged from 20 to 44% of total hemoglobin and approximately two-thirds of circulating red blood cells expressed HbF detected by immunofluorescence (F-cells). Gene therapy treatment of SCD mice ameliorated anemia, reduced hyperleukocytosis, improved renal function, and reduced iron accumulation in liver, spleen, and kidneys. Thus, modest levels of chimerism with donor cells expressing high levels of HbF from an insulated γ-globin lentiviral vector can improve the pathology of SCD in mice, thereby illustrating a potentially safe and effective strategy for gene therapy in humans.
RESUMEN
The kindlin/fermitin family includes three proteins involved in regulating integrin ligand-binding activity and adhesion. Loss-of-function mutations in kindlins1 and 3 have been implicated in Kindler Syndrome and Leukocyte Adhesion Deficiency III (LAD-III) respectively, whereas kindlin2 null mice are embryonic lethal. Post translational regulation of cell-cell and cell-ECM adhesion has long been presumed to be important for morphogenesis, however, few specific examples of activation-dependent changes in adhesion molecule function in normal development have been reported. In this study, antisense morpholinos were used to reduce expression of individual kindlins in Xenopus laevis embryos in order to investigate their roles in early development. Kindlin1 knockdown resulted in developmental delays, gross malformations of the gut and eventual lethality by tadpole stages. Kindlin2 morphant embryos displayed late stage defects in vascular maintenance and angiogenic branching consistent with kindlin2 loss of function in the mouse. Antisense morpholinos were also used to deplete maternal kindlin2 protein in oocytes and eggs. Embryos lacking maternal kindlin2 arrested at early cleavage stages due to failures in cytokinesis. Kindlin3 morphant phenotypes included defects in epidermal ciliary beating and partial paralysis at tailbud stages but these embryos recovered eventually as morpholino levels decayed. These results indicate a remarkably diverse range of kindlin functions in vertebrate development.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Animales , Animales Modificados Genéticamente , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Morfolinos/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neovascularización Fisiológica/genética , Oligorribonucleótidos Antisentido/genética , Oocitos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Xenopus/deficiencia , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
Here we report the cloning and functional characterization of the cyclin D-dependent kinase 4 and 6 (Cdk4/6) inhibitory protein Cdkn2d/p19Ink4d of Xenopuslaevis (Xl-Ink4d). Xl-Ink4d is the only Ink4 family gene highly expressed during Xenopus development and its transcripts were detected maternally and during neurulation. The Xl-Ink4d protein has 63% identity to mouse and human Cdkn2d/p19Ink4d and its function as a negative regulator of cell cycle traverse is evolutionary conserved. Indeed, Xl-lnk4d can functionally substitute for mouse Cdkn2d in binding to mouse Cdk4 and inhibiting cyclin-D1-dependent CDK4 kinase activity. Further, enforced expression of Xl-lnk4d arrests mouse fibroblasts in the G1 phase of the cell cycle. These findings indicate that CDKN2d/p19Ink4d is conserved through vertebrate evolution and suggest Xl-lnk4d may contribute to the development of Xenopuslaevis.
RESUMEN
The class II DNA "cut-and-paste" transposons have been used to efficiently modify the Xenopus genome for transgenesis applications. Once integrated, the transposon is an effective substrate for excision and re-integration (remobilization) elsewhere in the genome by simply supplying the transposase enzyme in trans. We have used two methods to remobilize transposons resident in the frog genome: micro-injection of transposase mRNA at the one-cell stage and expression of the enzyme in the germline from a transgene. Double-transgenic frogs (hoppers) that harbor transgenes for both the substrate transposon and the transposase enzyme are outcrossed to wild-type animals and the progeny are scored for changes in reporter gene expression. Although both methods work effectively to remobilize transposons, the breeding-mediated strategy eliminates the time-consuming micro-injection step; novel integration events are produced by simply outcrossing the hopper frogs. As each outcross of Xenopus tropicalis typically produces 2,000, or more, progeny, this method can be used to perform large-scale insertional mutagenesis screens in this highly tractable developmental model system.
Asunto(s)
Elementos Transponibles de ADN/genética , Pruebas Genéticas/métodos , Mutagénesis Insercional , Xenopus/genética , Crianza de Animales Domésticos , Sistemas de Identificación Animal , Animales , Animales Modificados Genéticamente , Cruzamiento , ADN/aislamiento & purificación , Femenino , Genoma , Técnicas de Genotipaje , Larva/genética , Masculino , Piel/químicaRESUMEN
The generation of transgenic animals is an essential tool for many genetic strategies. DNA "cut-and-paste" transposon systems can be used to efficiently modify the Xenopus genome. The DNA transposon substrate, harbored on a circularized plasmid, is co-injected into fertilized Xenopus embryos at the one-cell stage together with mRNA encoding the cognate transposase enzyme. The cellular machinery rapidly translates the exogenous mRNA to produce active transposase enzyme that catalyzes excision of the transposon substrate from the plasmid and stable integration into the genomic DNA.
Asunto(s)
Elementos Transponibles de ADN/genética , Mutagénesis Insercional , Xenopus/genética , Alimentación Animal , Crianza de Animales Domésticos , Animales , Animales Modificados Genéticamente/genética , Gonadotropina Coriónica/administración & dosificación , Técnicas de Cultivo , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Femenino , Masculino , Microinyecciones , Plásmidos , ARN Mensajero/genética , Sustancias para el Control de la Reproducción/administración & dosificaciónRESUMEN
BACKGROUND: The Sleeping Beauty (SB) transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization. RESULTS: Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'. CONCLUSIONS: In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.