RESUMEN
We report the safety and tolerability of 87 infusions of lentiviral vectormodified autologous CD4 T cells (VRX496-T; trade name, Lexgenleucel-T) in 17 HIV patients with well-controlled viremia. Antiviral effects were studied during analytic treatment interruption in a subset of 13 patients. VRX496-T was associated with a decrease in viral load set points in 6 of 8 subjects (P = .08). In addition, A â G transitions were enriched in HIV sequences after infusion, which is consistent with a model in which transduced CD4 T cells exert antisense-mediated genetic pressure on HIV during infection. Engraftment of vector-modified CD4 T cells was measured in gut-associated lymphoid tissue and was correlated with engraftment in blood. The engraftment half-life in the blood was approximately 5 weeks, with stable persistence in some patients for up to 5 years. Conditional replication of VRX496 was detected periodically through 1 year after infusion. No evidence of clonal selection of lentiviral vectortransduced T cells or integration enrichment near oncogenes was detected. This is the first demonstration that gene-modified cells can exert genetic pressure on HIV. We conclude that gene-modified T cells have the potential to decrease the fitness of HIV-1 and conditionally replicative lentiviral vectors have a promising safety profile in T cells.
Asunto(s)
Linfocitos T CD4-Positivos/trasplante , Terapia Genética/métodos , Infecciones por VIH/terapia , VIH-1/genética , Lentivirus/genética , Oligonucleótidos Antisentido/farmacología , Traslado Adoptivo/métodos , Adulto , Antivirales/efectos adversos , Antivirales/metabolismo , Antivirales/farmacología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/fisiología , Femenino , Terapia Genética/efectos adversos , Vectores Genéticos/efectos adversos , Vectores Genéticos/metabolismo , Vectores Genéticos/farmacología , Infecciones por VIH/genética , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Lentivirus/metabolismo , Lentivirus/fisiología , Masculino , Persona de Mediana Edad , Oligonucleótidos/administración & dosificación , Oligonucleótidos/genética , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/efectos adversos , Oligonucleótidos Antisentido/genética , Transducción Genética/métodos , Carga Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
PURPOSE: In spite of increased rates of complete response to initial chemotherapy, most patients with advanced ovarian cancer relapse and succumb to progressive disease. Immunotherapy may have potential for consolidation therapy. EXPERIMENTAL DESIGN: This randomized open-label phase I/II trial evaluated responses of patients with advanced ovarian cancer in remission for vaccination with monocyte-derived dendritic cells (DC) loaded with Her2/neu, hTERT, and PADRE peptides, with or without low-dose intravenous cyclophosphamide. All patients also received pneumococcal vaccine and were randomized to cyclophosphamide 2 days prior to first vaccination. Blood samples were analyzed by ELISPOT and flow cytometry. RESULTS: Of 11 patients, 2 recurred during vaccination. Nine received all 4 doses: 3 patients recurred at 6, 17, and 26 months, respectively, and 6 have no evidence of disease at 36 months. No grade 3/4 vaccine-related toxicities were noted. The 3-year overall survival was 90%. Patients receiving cyclophosphamide showed a non-significant improvement in survival over controls. Patients receiving cyclophosphamide had a transient reduction in neutrophils, but no change in total lymphocytes or regulatory T cells. Modest T-cell responses to Her2/neu and hTERT were seen post-vaccine by IFN-γ ELISPOT. Patients demonstrated below normal responses to the diphtheria conjugate protein CRM197, a component of the pneumococcal vaccine. CONCLUSIONS: In this setting, peptide-loaded DC vaccination elicits modest immune responses, but survival is promising. Pneumococcal vaccination revealed substantial immune suppression, even in patients in remission. Rational design of consolidative strategies for ovarian cancer will need to overcome tolerance and immunosuppression.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Ciclofosfamida/uso terapéutico , Células Dendríticas/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/terapia , Adolescente , Adulto , Proteínas Bacterianas/inmunología , Vacunas contra el Cáncer/inmunología , Terapia Combinada , Sinergismo Farmacológico , Femenino , Humanos , Interferón gamma/inmunología , Vacunas contra la Malaria/inmunología , Persona de Mediana Edad , Monocitos/inmunología , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/prevención & control , Neutrófilos/inmunología , Fragmentos de Péptidos/inmunología , Vacunas Neumococicas/inmunología , Vacunas Neumococicas/uso terapéutico , Receptor ErbB-2/inmunología , Tasa de Supervivencia , Linfocitos T Reguladores/inmunología , Adulto JovenRESUMEN
The uncoupling protein 2, UCP2, is a member of a family of inner mitochondrial membrane ion carriers involved in a host of metabolic processes. UCP2 protein is encoded by nuclear genome, but the protein is found exclusively in the mitochondria. The heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA-binding protein involved in many processes that compose gene expression, including mRNA processing and translation. The yeast three-hybrid screen revealed K protein bound to ucp2 mRNA through sites located in the 3'-untranslated region of the transcript. ucp2 mRNA-K protein complexes were associated with polysome-coated mitochondria. Expression of exogenous K protein augmented the insulin-induced mitochondrial level of UCP2 protein that was not accompanied by a corresponding increase in ucp2 mRNA. These results suggest the insulin stimulates translation of ucp2 mRNA in a process that involves K protein.
Asunto(s)
Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo K/fisiología , Insulina/farmacología , Proteínas de Transporte de Membrana/genética , Mitocondrias Hepáticas/química , Proteínas Mitocondriales/genética , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Sitios de Unión , Fraccionamiento Celular , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Humanos , Canales Iónicos , Neoplasias Hepáticas Experimentales , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/química , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/química , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae , Alineación de Secuencia , Transfección , Células Tumorales Cultivadas , Técnicas del Sistema de Dos Híbridos , Proteína Desacopladora 2RESUMEN
The heterogeneous nuclear ribonucleoprotein K protein is an RNA- and DNA-binding protein implicated in the regulation of multiple processes that comprise gene expression. We used chromatin immunoprecipitation (ChIP) assays to explore K protein interactions with serum-inducible, constitutively expressed and untranscribed gene loci in vivo. In the rat HTC-IR hepatoma cell line, serum treatment induced transient increases in the mRNA levels of two immediate-early genes, egr-1 and c-myc. ChIP analysis showed that the induction of egr-1 and c-myc genes was associated with a transient recruitment of K protein to multiple sites within each of these loci, including the promoter and transcribed regions. In contrast, recruitment of K protein to the constitutively transcribed beta-actin locus and to randomly chosen non-transcribed loci was far weaker. In rat mesangial cells, c-myc was constitutively expressed while egr-1 remained serum responsive. In these cells, ChIP analysis showed serum-induced recruitment to the inducible egr-1 but not to the c-myc locus. Pre-treatment with the transcription inhibitor actinomycin D blocked the inducible but not the constitutive binding of K protein to these loci. Taken together, the results of this study suggest that the transient recruitment of K protein to serum-responsive loci depends on the inducible transcription of these immediate-early genes.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Transcripción Genética/genética , Animales , Medio de Cultivo Libre de Suero/farmacología , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Expresión Génica , Proteínas Inmediatas-Precoces/genética , Pruebas de Precipitina/métodos , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Laminin is a multifunctional heterotrimeric protein present in extracellular matrix where it regulates processes that compose tissue architecture including cell differentiation. Laminin gamma1 is the most widely expressed laminin chain and its absence causes early lethality in mouse embryos. Laminin gamma1 chain gene (LAMC1) promoter contains several GC/GT-rich motifs including the bcn-1 element. Using the bcn-1 element as a bait in the yeast one-hybrid screen, we cloned the gut-enriched Kruppel-like factor (GKLF or KLF4) from a rat mesangial cell library. We show that GKLF binds bcn-1, but this binding is not required for the GKLF-mediated activation of the LAMC1 promoter. The activity of GKLF is dependent on a synergism with another Kruppel-like factor, Sp1. The LAMC1 promoter appears to have multiple GKLF- and Sp1-responsive elements which may account for the synergistic activation. We provide evidence that the synergistic action of GKLF and Sp1 is dependent on the promoter context and the integrity of GKLF activation and DNA-binding domain. GKLF is thought to participate in the switch from cell proliferation to differentiation. Thus, the Sp1-GKLF synergistic activation of the LAMC1 promoter may be one of the avenues for expression of laminin gamma1 chain when laminin is needed to regulate cell differentiation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Laminina/genética , Regiones Promotoras Genéticas/genética , Factor de Transcripción Sp1/metabolismo , Estómago/química , Factores de Transcripción/metabolismo , Activación Transcripcional , Secuencia de Aminoácidos , Animales , Sitios de Unión , Diferenciación Celular , Línea Celular , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Drosophila melanogaster , Células HeLa , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta/genética , Factor de Transcripción Sp1/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Dedos de ZincRESUMEN
Laminin is a major component of the extracellular matrix. The laminin gamma1 chain is the least variant component of the laminin heterotrimeric assembly. The laminin gamma1 chain gene (LAMC1) expression is induced by several factors, including transforming growth factor-beta (TGF-beta). LAMC1 promoter contains a highly conserved transcriptional element, bcn-1. We screened cDNA libraries with the yeast one-hybrid system to identify transcriptional factors that are recognized by the bcn-1 motif. Using this strategy we isolated the basic helix-loop-helix/leucine zipper (bHLHzip) E-box-binding transcription factor, TFE3. Until now, the E-box was the only element known to recruit the bHLHzip transcription factors. Although the bcn-1 element only remotely resembles the E-box sequence, we show that TFE3 binds and activates the bcn-1 element. TFE3 cooperates with Smad proteins in the activation of the LAMC1 promoter in cells, an effect that is critically dependent not only on the bcn-1 element but also on one of the Smad-binding elements (SBE). The cooperative induction of the LAMC1 promoter and the endogenous LAMC1 gene by TFE3 and Smad3 is augmented by the TGF-beta signaling pathway. Thus, the bcn-1 is a novel TFE3-dependent TGF-beta target element that regulates LAMC1 gene expression.