RESUMEN
The ß-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of ß-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound ß(E)/ß(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The ß(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated ß(E)-globin with partial instability. When this is compounded with a non-functional ß(0) allele, a profound decrease in ß-globin synthesis results, and approximately half of ß(E)/ß(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral ß-globin gene transfer, an adult patient with severe ß(E)/ß(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded ß-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.
Asunto(s)
Transfusión Sanguínea , Terapia Genética , Proteína HMGA2/metabolismo , Globinas beta/genética , Globinas beta/metabolismo , Talasemia beta/genética , Talasemia beta/terapia , Adolescente , Células Sanguíneas/citología , Células Sanguíneas/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Preescolar , Células Clonales/metabolismo , Expresión Génica , Vectores Genéticos/genética , Proteína HMGA2/genética , Homeostasis , Humanos , Lentivirus/genética , Masculino , MicroARNs/genética , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Factores de Tiempo , Activación Transcripcional , Adulto Joven , Talasemia beta/metabolismoRESUMEN
Retroviral vectors have been considered the most promising vehicles for the transfer of therapeutic genes into cells of the hematopoietic system. In clinical studies, however, cases of leukemoid clonogenic expansion of the transduced cells in vivo caused by semirandom integration of the foreign DNA in the genome have changed the focus to other types of vectors. In addition to their superior safety profile, a higher packaging capacity might be an advantage of non-viral vectors in certain applications. Prolonged transgene expression of non-viral vectors can be achieved by inclusion of Epstein-Barr virus (EBV)-derived elements mediating episomal replication and retention. Furthermore, a variety of cis acting elements have been explored in an attempt to enhance gene transfer efficiency. Our study confirmed that prolonged transgene expression can be achieved in B-lymphoid cells with EBV-derived vectors containing the EBV latent gene EBNA-1 and the EBV latent origin of replication oriP. In addition, we demonstrated that the inclusion of enhancer elements of the immunoglobulin κ light chain (Ei and E3') associated with its matrix attachment region (MAR) resulted in a 10-fold increase in transgene expression in B-lymphoid cells. It can be concluded that these elements are generally useful modules for improving the efficiencies of non-viral vectors in the B-lymphoid lineage.
RESUMEN
Mutator phenotypes, a common and largely unexplained attribute of human cancer, might be better understood in mouse tumors containing reporter genes for accurate mutation enumeration and analysis. Previous work on peritoneal plasmacytomas (PCTs) in mice suggested that PCTs have a mutator phenotype caused by Myc-deregulating chromosomal translocations and/or phagocyte-induced mutagenesis due to chronic inflammation. To investigate this hypothesis, we generated PCTs that harbored the transgenic shuttle vector, pUR288, with a lacZ reporter gene for the assessment of mutations in vivo. PCTs exhibited a 5.5 times higher mutant frequency in lacZ (40.3 +/- 5.1 x 10(-5)) than in normal B cells (7.36 +/- 0.77 x 10(-5)), demonstrating that the tumors exhibit the phenotype of increased mutability. Studies on lacZ mutant frequency in serially transplanted PCTs and phagocyte-induced lacZ mutations in B cells in vitro indicated that mutant levels in tumors are not determined by exogenous damage inflicted by inflammatory cells. In vitro studies with a newly developed transgenic model of inducible Myc expression (Tet-off/MYC) showed that deregulated Myc sensitizes B cells to chemically induced mutations, but does not cause, on its own, mutations in lacZ. These findings suggested that the hypermutability of PCT is governed mainly by intrinsic features of tumor cells, not by deregulated Myc or chronic inflammation.
Asunto(s)
Genes myc/genética , Inflamación/complicaciones , Mutagénesis/genética , Plasmacitoma/genética , beta-Galactosidasa/genética , Animales , Linfocitos B/fisiología , Genes Reporteros , Predisposición Genética a la Enfermedad/genética , Vectores Genéticos , Cinética , Ratones , Ratones Transgénicos , Plasmacitoma/etiología , Plasmacitoma/patologíaRESUMEN
MHC class I ligands are recruited from the cytosolic peptide pool, whose size is likely to depend on the balance between peptide generation by the proteasome and peptide degradation by downstream peptidases. We asked what fraction of this pool is available for presentation, and how the size of this fraction is modulated by peptide affinity for the TAP transporters. A model epitope restricted by HLA-A2 and a series of epitope precursors with N-terminal extensions by single residues modifying TAP affinity were expressed in a system that allowed us to monitor and modulate cytosolic peptide copy numbers. We show that presentation varies strongly according to TAP affinities of the epitope precursors. The fraction of cytosolic peptides recruited for MHC presentation does not exceed 1% and is more than two logs lower for peptides with very low TAP affinities. Therefore, TAP affinity has a substantial impact on MHC class I Ag presentation.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Transportadoras de Casetes de Unión a ATP/metabolismo , Presentación de Antígeno , Antígenos H-2/metabolismo , Antígeno HLA-A2/metabolismo , Péptidos/inmunología , Péptidos/metabolismo , Animales , Presentación de Antígeno/genética , Unión Competitiva/genética , Unión Competitiva/inmunología , Línea Celular Transformada , Citosol/inmunología , Citosol/metabolismo , Pruebas Inmunológicas de Citotoxicidad/métodos , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Proteínas Fluorescentes Verdes , Antígenos H-2/inmunología , Antígeno HLA-A2/inmunología , Antígeno de Histocompatibilidad H-2D , Humanos , Proteínas Luminiscentes/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Péptidos/genética , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Transfección/métodos , Células Tumorales CultivadasRESUMEN
To characterize the effects of inhibitors of Epstein-Barr virus (EBV) reactivation, we established Raji DR-LUC cells as a new test system. These cells contain the firefly luciferase (LUC) gene under the control of an immediate-early gene promoter (duplicated right region [DR]) of EBV on a self-replicating episome. Luciferase induction thus serves as an intrinsic marker indicative for EBV reactivation from latency. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the viral key activator BamH fragment Z left frame 1 (BZLF1) protein ("ZEBRA") in this system, as demonstrated by induction of the BZLF1 protein-responsive DR promoter upstream of the luciferase gene. Conversely, both BZLF1 protein and luciferase induction were inhibited effectively by the chemopreventive agent curcumin. Semiquantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) further demonstrated that the EBV inducers TPA, sodium butyrate, and transforming growth factor-beta (TGF-beta) increased levels of the mRNA of BZLF1 mRNA at 12, 24, and 48 h after treatment in these cells. TPA treatment also induced luciferase mRNA with similar kinetics. Curcumin was found to be highly effective in decreasing TPA-, butyrate-, and TGF-beta-induced levels of BZLF1 mRNA, and of TPA-induced luciferase mRNA, indicating that three major pathways of EBV are inhibited by curcumin. Electrophoretic mobility shift assays (EMSA) showed that activator protein 1 (AP-1) binding to a cognate AP-1 sequence was detected at 6 h and could be blocked by curcumin. Protein binding to the complete BZLF1 promoter ZIII site (ZIIIA+ZIIIB) demonstrated several specific complexes that gave weak signals at 6 h and 12 h but strong signals at 24 h, all of which were reduced after application of curcumin. Autostimulation of BZLF1 mRNA induction through binding to the ZIII site at 24 h was confirmed by antibody-induced supershift analysis. The present results confirm our previous finding that curcumin is an effective agent for inhibition of EBV reactivation in Raji DR-CAT cells (carrying DR-dependent chloramphenicol acetyltransferase), and they show for the first time that curcumin inhibits EBV reactivation mainly through inhibition of BZLF1 gene transcription.
Asunto(s)
Anticarcinógenos/farmacología , Curcumina/farmacología , Proteínas de Unión al ADN/genética , Regulación Viral de la Expresión Génica/efectos de los fármacos , Herpesvirus Humano 4/efectos de los fármacos , Transactivadores/genética , Proteínas Virales , Carcinógenos/antagonistas & inhibidores , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Cinética , Luciferasas/análisis , Luciferasas/genética , Regiones Promotoras Genéticas , ARN Viral/biosíntesis , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Transactivadores/biosíntesis , Factores de Transcripción/metabolismo , Activación Transcripcional/efectos de los fármacos , Células Tumorales Cultivadas , Latencia del VirusRESUMEN
Lymphoblastoid cell lines, generated by immortalization of normal B cells by Epstein-Barr virus (EBV) in vitro, have strong antigen-presenting capacity, are sensitive to EBV-specific cytotoxic T cells, and are highly allostimulatory in mixed lymphocyte culture. By contrast, EBV-positive Burkitt lymphoma (BL) cells are poor antigen presenters, are not recognized by EBV-specific cytotoxic T cells, and are poorly allostimulatory, which raises the question of whether immunological pressure exerted during BL pathogenesis in vivo has selected for a 'nonimmunogenic' tumor phenotype. The present work addresses this question by examining the immunogenicity/antigenicity of cell lines, generated by conversion of a conditionally immortalized lymphoblastoid cell line to permanent growth independent of EBV-latent proteins by introduction of a constitutively active or tetracycline-regulated c-myc gene (A1 and P493-6 cells, respectively). Compared with its parental lymphoblastoid cell line, A1 cells showed many of the features of the nonimmunogenic BL phenotype, namely poor allostimulatory activity, poor antigen-presenting function associated with impaired proteasomal activity, down-regulation of peptide transporter, reduced HLA class I expression, and an inability to present endogenously expressed EBV-latent proteins to cytotoxic T cells. P493-6 cells, when grown in the presence of estrogen with the exogenous c-myc gene switched off, were strongly immunogenic. The cells had lost their immunogenic potential, however, when grown on a c-myc-driven proliferation program in the absence of estrogen. Deregulation of c-myc, a step central to the development of uncontrolled BL cell growth in vivo, can thus impose a nonimmunogenic phenotype on proliferating human B cells in the absence of any immune pressure.
Asunto(s)
Linfocitos B/inmunología , Herpesvirus Humano 4/inmunología , Proteínas Proto-Oncogénicas c-myc/fisiología , Presentación de Antígeno , Linfocitos B/virología , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Vectores Genéticos , Antígenos HLA-A/análisis , Antígeno HLA-A11 , Antígeno HLA-B7/análisis , Humanos , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Fenotipo , Linfocitos T Citotóxicos/inmunología , Virus Vaccinia/genética , Proteínas ViralesRESUMEN
Epstein-Barr virus (EBV) transforms primary B cells in vitro. Established cell lines adopt a lymphoblastoid phenotype (LCL). In contrast, EBV-positive Burkitt's lymphoma (BL) cells, in which the proto-oncogene c-myc is constitutively activated, do not express a lymphoblastoid phenotype in vivo. The two different phenotypes are paralleled by two distinct programmes of EBV latent gene expression termed latency type I in BL cells and type III in LCL. Human B cell lines were established from a conditional LCL (EREB2-5) by overexpression of c-myc and inactivation of EBV nuclear protein 2 (EBNA2). These cells (A1 and P493-6) adopted a BL phenotype in the absence of EBNA2. However, the EBV latency I promoter Qp was not activated. Instead, the latency III promoter Cp remained active. These data suggest that the induction of a BL phenotype by overexpression of c-myc in an LCL is not necessarily paralleled by an EBV latency III-to-I switch.