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Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.
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Receptores Acoplados a Proteínas G , Fosforilación , Ligandos , Receptores Acoplados a Proteínas G/metabolismo , InmunoensayoRESUMEN
Blockade of the inhibitory receptor TIM-3 shows efficacy in cancer immunotherapy clinical trials. TIM-3 inhibits production of the chemokine CXCL9 by XCR1+ classical dendritic cells (cDC1), thereby limiting antitumor immunity in mammary carcinomas. We found that increased CXCL9 expression by splenic cDC1s upon TIM-3 blockade required type I interferons and extracellular DNA. Chemokine expression as well as combinatorial efficacy of TIM-3 blockade and paclitaxel chemotherapy were impaired by deletion of Cgas and Sting. TIM-3 blockade increased uptake of extracellular DNA by cDC1 through an endocytic process that resulted in cytoplasmic localization. DNA uptake and efficacy of TIM-3 blockade required DNA binding by HMGB1, while galectin-9-induced cell surface clustering of TIM-3 was necessary for its suppressive function. Human peripheral blood cDC1s also took up extracellular DNA upon TIM-3 blockade. Thus, TIM-3 regulates endocytosis of extracellular DNA and activation of the cytoplasmic DNA sensing cGAS-STING pathway in cDC1s, with implications for understanding the mechanisms underlying TIM-3 immunotherapy.
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ADN/metabolismo , Células Dendríticas/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Transducción de Señal/fisiología , Animales , Transporte Biológico/fisiología , Línea Celular , Línea Celular Tumoral , Quimiocinas/metabolismo , Citoplasma/metabolismo , Endocitosis/fisiología , Femenino , Células HEK293 , Humanos , Inmunoterapia/métodos , Ratones , Ratones Endogámicos C57BLRESUMEN
PURPOSE: To report the long-term outcomes of neoadjuvant altered fractionation short-course radiotherapy in 271 consecutive patients with stage II-III rectal cancer. PATIENTS AND METHODS: This was a retrospective single institution study with median follow-up of 101 months (8.4 years). Patients who were alive at the time of analysis in 2018 were contacted to obtain functional outcome data (phone interview). Radiotherapy consisted of 25 Gy in 10 fractions of 2.5 Gy administered twice daily. Median time interval to surgery was 5 days. RESULTS: Local relapse was observed in 12 patients (4.4%) after a median of 28 months. Overall survival after 5 and 10 years was 73 and 55.5%, respectively (corresponding disease-free survival 65.5 and 51%). Of all patients without permanent stoma, 79% reported no low anterior resection syndrome (LARS; 0-20 points), 9% reported LARS with 21-29 points and 12% serious LARS (30-42 points). CONCLUSION: The present radiotherapy regimen was feasible and resulted in low rates of local relapse. Most patients reported good functional outcomes.
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Fraccionamiento de la Dosis de Radiación , Radioterapia Adyuvante/métodos , Neoplasias del Recto/radioterapia , Adulto , Anciano , Anciano de 80 o más Años , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante/métodos , Terapia Neoadyuvante/mortalidad , Recurrencia Local de Neoplasia/epidemiología , Radioterapia Adyuvante/mortalidad , Neoplasias del Recto/mortalidad , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Targeting neoantigens has become an attractive strategy for cancer immunotherapy. Epitope prediction algorithms facilitate rapid selection of potential neoantigens, but are plagued with high false-positive and false-negative rates. Here we review ex vivo technologies for biological identification of neoantigens to improve empirical prioritization for immunotherapy.
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Antígenos de Neoplasias/inmunología , Inmunoterapia , Neoplasias/inmunología , Neoplasias/terapia , HumanosRESUMEN
Background: Combined immunotherapy approaches are promising cancer treatments. We evaluated anti-programmed cell death protein 1 (PD-1) treatment combined with gene-mediated cytotoxic immunotherapy (GMCI) performed by intratumoral injection of a prodrug metabolizing nonreplicating adenovirus (AdV-tk), providing in situ chemotherapy and immune stimulation. Methods: The effects of GMCI on PD ligand 1 (PD-L1) expression in glioblastoma were investigated in vitro and in vivo. The efficacy of the combination was investigated in 2 syngeneic mouse glioblastoma models (GL261 and CT-2A). Immune infiltrates were analyzed by flow cytometry. Results: GMCI upregulated PD-L1 expression in vitro and in vivo. Both GMCI and anti-PD-1 increased intratumoral T-cell infiltration. A higher percentage of long-term survivors was observed in mice treated with combined GMCI/anti-PD-1 relative to single treatments. Long-term survivors were protected from tumor rechallenge, demonstrating durable memory antitumor immunity. GMCI led to elevated interferon gamma positive T cells and a lower proportion of exhausted double positive PD1+TIM+CD8+ T cells. GMCI also increased PD-L1 levels on tumor cells and infiltrating macrophages/microglia. Our data suggest that anti-PD-1 treatment improves the effectiveness of GMCI by overcoming interferon-induced PD-L1-mediated inhibitory signals, and GMCI improves anti-PD-1 efficacy by increasing tumor-infiltrating T-cell activation. Conclusions: Our data show that the GMCI/anti-PD-1 combination is well tolerated and effective in glioblastoma mouse models. These results support evaluation of this combination in glioblastoma patients.
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Anticuerpos Monoclonales/uso terapéutico , Neoplasias Encefálicas , Terapia Combinada , Glioblastoma , Inmunoterapia , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Terapia Combinada/métodos , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/inmunología , Humanos , Inmunoterapia/métodos , Ratones , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Immunotherapeutic vaccines have emerged as a novel treatment modality for genital herpes, a sexually transmitted disease mainly caused by herpes simplex virus type 2. The approaches to identify potential vaccine antigens have evolved from classic virus attenuation and characterization of antibody and T cell responses in exposed, but seronegative individuals, to systematic screens for novel T cell antigens. Combined with implementation of novel vaccine concepts revolving around immune evasion and local recruitment of immune effectors, the development of a safe and effective therapeutic vaccine is within reach. Here, we describe the vaccine approaches that currently show promise at clinical and pre-clinical stages and link them to the evolving scientific strategies that led to their identification.
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Herpes Genital/terapia , Vacunas contra el Virus del Herpes Simple/inmunología , Herpesvirus Humano 2/inmunología , Inmunoterapia/métodos , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Ensayos Clínicos como Asunto , Diseño de Fármacos , Descubrimiento de Drogas , Herpes Genital/inmunología , Herpes Genital/virología , Vacunas contra el Virus del Herpes Simple/administración & dosificación , Humanos , Evasión Inmune , Ratones , Linfocitos T/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Oncolytic viral (OV) therapy, which uses genetically engineered tumor-targeting viruses, is being increasingly used in cancer clinical trials due to the direct cytolytic effects of this treatment that appear to provoke a robust immune response against the tumor. As OVs enter tumor cells, intrinsic host defenses have the potential to hinder viral replication and spread within the tumor mass. In this report, we show that histone deacetylase 6 (HDAC6) in tumor cells appears to alter the trafficking of post-entry OVs from the nucleus toward lysosomes. In glioma cell lines and glioma-stem-like cells, HDAC6 inhibition (HDAC6i) by either pharmacologic or genetic means substantially increased replication of oncolytic herpes simplex virus type 1 (oHSV). Moreover, HDAC6i increased shuttling of post-entry oHSV to the nucleus. In addition, electron microscopic analysis revealed that post-entry oHSVs are preferentially taken up into glioma cells through the endosomal pathway rather than via fusion at the cell surface. Together, these findings illustrate a mechanism of glioma cell defense against an incoming infection by oHSV and identify possible approaches to enhance oHSV replication and subsequent lysis of tumor cells.
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Anilidas/farmacología , Neoplasias Encefálicas/virología , Glioma/virología , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/fisiología , Ácidos Hidroxámicos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Viroterapia Oncolítica/métodos , Virus Oncolíticos/fisiología , Replicación Viral/efectos de los fármacos , Acetilación , Acetiltransferasas/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Cápside/metabolismo , Línea Celular Tumoral , Núcleo Celular/virología , Endocitosis/efectos de los fármacos , Glioma/genética , Glioma/patología , Herpesvirus Humano 1/fisiología , Histona Desacetilasa 6 , Histona Desacetilasas/genética , Humanos , Técnicas In Vitro , Interferón beta/antagonistas & inhibidores , Interferón beta/farmacología , Lisosomas/virología , Proteínas de Microtúbulos , Microtúbulos/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , ARN Mensajero/genética , ARN Neoplásico/genética , ARN Interferente Pequeño/genética , ARN Viral/genética , Esferoides Celulares , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Ácido Valproico/farmacologíaRESUMEN
BACKGROUND: A phase I/II trial for glioblastoma with the oncolytic adenovirus Delta24-RGD was recently completed. Delta24-RGD conditionally replicates in cells with a disrupted retinoblastoma-pathway and enters cells via αvß3/5 integrins. Glioblastomas are differentially sensitive to Delta24-RGD. HDAC inhibitors (HDACi) affect integrins and share common cell death pathways with Delta24-RGD. We studied the combination treatment effects of HDACi and Delta24-RGD in patient-derived glioblastoma stem-like cells (GSC), and we determined the most effective HDACi. METHODS: SAHA, Valproic Acid, Scriptaid, MS275 and LBH589 were combined with Delta24-RGD in fourteen distinct GSCs. Synergy was determined by Chou Talalay method. Viral infection and replication were assessed using luciferase and GFP encoding vectors and hexon-titration assays. Coxsackie adenovirus receptor and αvß3 integrin levels were determined by flow cytometry. Oncolysis and mechanisms of cell death were studied by viability, caspase-3/7, LDH and LC3B/p62, phospho-p70S6K. Toxicity was studied on normal human astrocytes. MGMT promotor methylation status, TCGA classification, Rb-pathway and integrin gene expression levels were assessed as markers of responsiveness. RESULTS: Scriptaid and LBH589 acted synergistically with Delta24-RGD in approximately 50% of the GSCs. Both drugs moderately increased αvß3 integrin levels and viral infection in responding but not in non-responding GSCs. LBH589 moderately increased late viral gene expression, however, virus titration revealed diminished viral progeny production by both HDACi, Scriptaid augmented caspase-3/7 activity, LC3B conversion, p62 and phospho-p70S6K consumption, as well as LDH levels. LBH589 increased LDH and phospho-p70S6K consumption. Responsiveness correlated with expression of various Rb-pathway genes and integrins. Combination treatments induced limited toxicity to human astrocytes. CONCLUSION: LBH589 and Scriptaid combined with Delta24-RGD revealed synergistic anti-tumor activity in a subset of GSCs. Both HDACi moderately augmented viral infection and late gene expression, but slightly reduced progeny production. The drugs differentially activated multiple cell death pathways. The limited toxicity on astrocytes supports further evaluation of the proposed combination therapies.
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Neoplasias Encefálicas/patología , Glioblastoma/patología , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Hidroxilaminas/farmacología , Indoles/farmacología , Virus Oncolíticos , Quinolinas/farmacología , Adenoviridae/genética , Animales , Apoptosis/efectos de los fármacos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Autofagia/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Supervivencia Celular , Metilación de ADN , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Integrina alfaVbeta3/metabolismo , Ratones , Mutación , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Viroterapia Oncolítica , Panobinostat , Regiones Promotoras Genéticas , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Replicación Viral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as immune-checkpoint-inhibiting antibodies or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno-virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a panel of melanoma cell lines and animal models, and summarized the melanoma-specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK-MEL-28 melanoma xenografts in nude mice when combined with low-dose cyclophosphamide. Furthermore, virally-expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. In ATAP, treatments appeared safe and well-tolerated. Four out of nine melanoma patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, two had stable disease, and one had progressive disease. Two patients were alive at 559 and 2,149 days after treatment. Ad5/3-D24-GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Melanoma/terapia , Viroterapia Oncolítica/métodos , Adenoviridae/genética , Animales , Diferenciación Celular/fisiología , Línea Celular Tumoral , Cricetinae , Ciclofosfamida/farmacología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Macrófagos/patología , Macrófagos/virología , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/virología , Ratones , Ratones Desnudos , Monocitos/patología , Monocitos/virología , Distribución Aleatoria , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody therapy of solid cancers is well established, but suffers from unsatisfactory tumor penetration of large immunoglobulins or from low serum retention of antibody fragments. Oncolytic viruses are in advanced clinical development showing excellent safety, but suboptimal potency due to limited virus spread within tumors. Here, by developing an immunoRNase-encoding oncolytic adenovirus, we combine viral oncolysis with intratumoral genetic delivery of a small antibody-fusion protein for targeted bystander killing of tumor cells (viro-antibody therapy). Specifically, we explore genetic delivery of a small immunoRNase consisting of an EGFR-binding scFv antibody fragment fused to the RNase Onconase (ONC(EGFR)) that induces tumor cell death by RNA degradation after cellular internalization. Onconase is a frog RNase that combines lack of immunogenicity and excellent safety in patients with high tumor killing potency due to its resistance to the human cytosolic RNase inhibitor. We show that ONC(EGFR) expression by oncolytic adenoviruses is feasible with an optimized, replication-dependent gene expression strategy. Virus-encoded ONC(EGFR) induces potent and EGFR-dependent bystander killing of tumor cells. Importantly, the ONC(EGFR)-encoding oncolytic adenovirus showed dramatically increased cytotoxicity specifically to EGFR-positive tumor cells in vitro and significantly enhanced therapeutic activity in a mouse xenograft tumor model. The latter demonstrates that ONC(EGFR) is expressed at levels sufficient to trigger tumor cell killing in vivo. The established ONC(EGFR)-encoding oncolytic adenovirus represents a novel agent for treatment of EGFR-positive tumors. This viro-antibody therapy platform can be further developed for targeted/personalized cancer therapy by exploiting antibody diversity to target further established or emerging tumor markers or combinations thereof.
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Adenoviridae/genética , Antineoplásicos/administración & dosificación , Antineoplásicos/metabolismo , Vectores Genéticos/genética , Virus Oncolíticos/genética , Ribonucleasas/administración & dosificación , Ribonucleasas/metabolismo , Animales , Anticuerpos Antivirales , Línea Celular Tumoral , Receptores ErbB/metabolismo , Femenino , Terapia Genética/métodos , Humanos , Ratones , Ratones Endogámicos BALB C , Viroterapia Oncolítica/métodos , ARN/metabolismo , Replicación Viral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We hypothesized that the combination of oncolytic virotherapy with immune checkpoint modulators would reduce tumor burden by direct cell lysis and stimulate antitumor immunity. In this study, we have generated attenuated Measles virus (MV) vectors encoding antibodies against CTLA-4 and PD-L1 (MV-aCTLA-4 and MV-aPD-L1). We characterized the vectors in terms of growth kinetics, antibody expression, and cytotoxicity in vitro. Immunotherapeutic effects were assessed in a newly established, fully immunocompetent murine model of malignant melanoma, B16-CD20. Analyses of tumor-infiltrating lymphocytes and restimulation experiments indicated a favorable immune profile after MV-mediated checkpoint modulation. Therapeutic benefits in terms of delayed tumor progression and prolonged median overall survival were observed for animals treated with vectors encoding anti-CTLA-4 and anti-PD-L1, respectively. Combining systemic administration of antibodies with MV treatment also improved therapeutic outcome. In vivo oncolytic efficacy against human tumors was studied in melanoma xenografts. MV-aCTLA-4 and MV-aPD-L1 were equally efficient as parental MV in this model, with high rates of complete tumor remission (> 80%). Furthermore, we could demonstrate lysis of tumor cells and transgene expression in primary tissue from melanoma patients. The current results suggest rapid translation of combining immune checkpoint modulation with oncolytic viruses into clinical application.
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Antígeno B7-H1/metabolismo , Antígeno CTLA-4/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma Experimental/terapia , Virus Oncolíticos/inmunología , Animales , Vectores Genéticos/administración & dosificación , Virus del Sarampión/genética , Virus del Sarampión/inmunología , Virus del Sarampión/metabolismo , Melanoma Experimental/inmunología , Ratones , Viroterapia Oncolítica , Virus Oncolíticos/genética , Virus Oncolíticos/metabolismo , Resultado del TratamientoRESUMEN
Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK). This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis.
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Adenoviridae/metabolismo , Receptor EphA2/metabolismo , Animales , Línea Celular , Femenino , Humanos , Melanoma/metabolismo , Melanoma/terapia , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite extensive research, current glioma therapies are still unsatisfactory, and novel approaches are pressingly needed. In recent years, both nonreplicative viral vectors and replicating oncolytic viruses have been developed for brain cancer treatment, and the mechanistic background of their cytotoxicity has been unveiled. A growing number of clinical trials have convincingly established viral therapies to be safe in glioma patients, and maximum tolerated doses have generally not been reached. However, evidence for therapeutic benefit has been limited: new generations of therapeutic vectors need to be developed in order to target not only tumor cells but also the complex surrounding microenvironment. Such therapies could also direct long-lasting immune responses toward the tumor while reducing early antiviral reactions. Furthermore, viral delivery methods are to be improved and viral spread within the tumor will have to be enhanced. Here, we will review the outcome of completed glioma virus therapy trials as well as highlight the ongoing clinical activities. On this basis, we will give an overview of the numerous strategies to enhance therapeutic efficacy of new-generation viruses and novel treatment regimens. Finally, we will conclude with approaches that may be crucial to the development of successful glioma therapies in the future.
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Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Viroterapia Oncolítica , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/virología , Terapia Genética , Glioblastoma/genética , Glioblastoma/virología , Humanos , Investigación Biomédica TraslacionalRESUMEN
Aptazymes are small, ligand-dependent self-cleaving ribozymes that function independently of transcription factors and can be customized for induction by various small molecules. Here, we introduce these artificial riboswitches for regulation of DNA and RNA viruses. We hypothesize that they represent universally applicable tools for studying viral gene functions and for applications as a safety switch for oncolytic and live vaccine viruses. Our study shows that the insertion of artificial aptazymes into the adenoviral immediate early gene E1A enables small-molecule-triggered, dose-dependent inhibition of gene expression. Aptazyme-mediated shutdown of E1A expression translates into inhibition of adenoviral genome replication, infectious particle production, and cytotoxicity/oncolysis. These results provide proof of concept for the aptazyme approach for effective control of biological outcomes in eukaryotic systems, specifically in virus infections. Importantly, we also demonstrate aptazyme-dependent regulation of measles virus fusion protein expression, translating into potent reduction of progeny infectivity and virus spread. This not only establishes functionality of aptazymes in fully cytoplasmic genetic systems, but also implicates general feasibility of this strategy for application in viruses with either DNA or RNA genomes. Our study implies that gene regulation by artificial riboswitches may be an appealing alternative to Tet- and other protein-dependent gene regulation systems, based on their small size, RNA-intrinsic mode of action, and flexibility of the inducing molecule. Future applications range from gene analysis in basic research to medicine, for example as a safety switch for new generations of efficiency-enhanced oncolytic viruses.
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Virus ADN/genética , Virus ADN/fisiología , Regulación Viral de la Expresión Génica , Virus ARN/genética , Virus ARN/fisiología , Riboswitch/genética , Replicación Viral/genética , Adenoviridae/genética , Adenoviridae/patogenicidad , Adenoviridae/fisiología , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Línea Celular , Virus ADN/patogenicidad , Genes Virales/genética , Ligandos , Virus del Sarampión/genética , Virus del Sarampión/patogenicidad , Virus del Sarampión/fisiología , Virus Oncolíticos/genética , Virus Oncolíticos/fisiología , Virus ARN/patogenicidad , ARN Catalítico/metabolismo , Virión/fisiología , Internalización del VirusRESUMEN
Effective treatment modalities for advanced melanoma are desperately needed. An innovative approach is virotherapy, in which viruses are engineered to infect cancer cells, resulting in tumor cell lysis and an amplification effect by viral replication and spread. Ideally, tumor selectivity of these oncolytic viruses is already determined during viral cell binding and entry, which has not been reported for melanoma. We engineered an oncolytic measles virus entering melanoma cells through the high molecular weight melanoma-associated antigen (HMWMAA) and proved highly specific infection and spread in melanoma cells. We further enhanced this oncolytic virus by inserting the FCU1 gene encoding the yeast-derived prodrug convertases cytosine deaminase and uracil phosphoribosyltransferase. Combination treatment with armed and retargeted MV-FCU1-αHMWMAA and the prodrug 5-fluorocytosine (5-FC) led to effective prodrug conversion to 5-fluorouracil, extensive cytotoxicity to melanoma cells, and excessive bystander killing of noninfected cells. Importantly, HMWMAA-retargeted MV showed antitumor activity in a human xenograft mouse model, which was further increased by the FCU1/5-FC prodrug activation system. Finally, we demonstrated susceptibility of melanoma skin metastasis biopsies to HMWMAA-retargeted MV. The highly selective, entry-targeted and armed oncolytic virus MV-FCU1-αHMWMAA may become a potent building block of future melanoma therapies.
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Virus del Sarampión/genética , Melanoma/tratamiento farmacológico , Viroterapia Oncolítica/métodos , Profármacos/farmacocinética , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Antígenos de Neoplasias/metabolismo , Antimetabolitos/metabolismo , Biopsia , Línea Celular Tumoral , Chlorocebus aethiops , Terapia Combinada , Femenino , Flucitosina/metabolismo , Ingeniería Genética/métodos , Genoma Viral/genética , Humanos , Melanoma/secundario , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Recombinantes/genética , Neoplasias Cutáneas/patología , Células Vero , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.
Asunto(s)
Adenoviridae/metabolismo , Virus Oncolíticos/metabolismo , Parvovirus/metabolismo , Animales , Secuencia de Bases , Proliferación Celular , Supervivencia Celular , Clonación Molecular , Fibroblastos/citología , Eliminación de Gen , Células HEK293 , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Sales de Tetrazolio/farmacología , Tiazoles/farmacología , Virología/métodosRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma(1), a highly vascularized tumor originating from lymphatic endothelial cells, and of at least two different B cell malignancies(2,3). A dimeric complex formed by the envelope glycoproteins H and L (gH-gL) is required for entry of herpesviruses into host cells(4). We show that the ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH-gL. EphA2 co-precipitated with both gH-gL and KSHV virions. Infection of human epithelial cells with a GFP-expressing recombinant KSHV strain, as measured by FACS analysis, was increased upon overexpression of EphA2. Antibodies against EphA(2) and siRNAs directed against EphA2 inhibited infection of endothelial cells. Pretreatment of KSHV with soluble EphA2 resulted in inhibition of KSHV infection by up to 90%. This marked reduction of KSHV infection was seen with all the different epithelial and endothelial cells used in this study. Similarly, pretreating epithelial or endothelial cells with the soluble EphA2 ligand ephrinA4 impaired KSHV infection. Deletion of the gene encoding EphA2 essentially abolished KSHV infection of mouse endothelial cells. Binding of gH-gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry(5,6). Quantitative RT-PCR and in situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from human Kaposi's sarcoma lesions or unaffected human lymphatic endothelium, and in situ in Kaposi's sarcoma specimens, respectively. Taken together, our results identify EphA2, a tyrosine kinase with known functions in neovascularization and oncogenesis, as an entry receptor for KSHV.
Asunto(s)
Herpesvirus Humano 8/fisiología , Receptor EphA2/fisiología , Receptores Virales/fisiología , Animales , Línea Celular , Endocitosis , Humanos , Ratones , Fosforilación , Proteínas del Envoltorio Viral/fisiología , Proteínas Virales/fisiologíaRESUMEN
Several challenges need to be addressed when developing viruses for clinical applications in gene therapy, vaccination, or viral oncolysis, including specific and efficient target cell transduction, virus delivery via the blood stream, and evasion of pre-existing immunity. With rising frequency, these goals are tackled by generating chimeric viruses containing nucleic acid fragments or proteins from two or more different viruses, thus combining different beneficial features of the parental viruses. These chimeras have boosted the development of virus-based treatment regimens for major inherited and acquired diseases, including cancer. Using adenoviruses as the paradigm and prominent examples from other virus families, we review the technological and functional advances in therapeutic virus chimera development and recent successful applications that can pave the way for future therapies.
Asunto(s)
Terapia Genética , Vectores Genéticos/genética , Neoplasias/terapia , Recombinación Genética , Virus/genética , Adenoviridae/genética , Animales , Virus ADN/genética , Vectores Genéticos/uso terapéutico , Humanos , Neoplasias/genética , Viroterapia Oncolítica , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds α(v)ß(3) and α(v)ß(5) integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing α(v)ß(5) integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.