RESUMEN
Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.
Asunto(s)
Oligonucleótidos/química , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/genética , ARN no Traducido/química , Imagen de Cuerpo Entero/métodos , Animales , Células HCT116 , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Ratones , Oligonucleótidos/farmacocinética , Oligonucleótidos/uso terapéutico , Especificidad de Órganos , ARN Mensajero/química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN no Traducido/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Distribución TisularRESUMEN
Sphingosine-1-phosphate (S1P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S1P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S1P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S1P1 and S1P3 receptors and demonstrate that FTY720P activates Gi protein-mediated signaling cascades. We also show that FTY720P as well as the S1P1-selective agonist SEW2871 stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S1P1 receptors, whereas S1P signals through both S1P1 and S1P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S1P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.
Asunto(s)
Astrocitos/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Inmunosupresores/farmacología , Glicoles de Propileno/farmacología , Receptores de Lisoesfingolípidos/fisiología , Esfingosina/análogos & derivados , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Animales , Astrocitos/fisiología , Señalización del Calcio/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Clorhidrato de Fingolimod , Proteína Ácida Fibrilar de la Glía/metabolismo , Ácido Glutámico/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Fosfatos de Inositol/metabolismo , Técnicas de Cultivo de Órganos , Oxadiazoles/farmacología , Ratas , Receptores de Lisoesfingolípidos/agonistas , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Esfingosina/farmacología , Tiofenos/farmacología , beta-Alanina/análogos & derivados , beta-Alanina/farmacologíaRESUMEN
Functional genomics is inundating the pharmaceutical industry with large numbers of potential gene targets from several sources such as gene expression profiling experiments (DNA microchips, proteomics) or database mining. Oligonucleotide-based RNA-knock down technologies such as antisense or RNA interference can aid in the filtering and prioritization of target candidates in the drug discovery process.
Asunto(s)
Oligorribonucleótidos/síntesis química , Industria Farmacéutica/métodos , Genómica , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/genética , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genéticaRESUMEN
Synthetic 21-bp-long short interfering RNAs (siRNA) can stimulate sequence-specific mRNA degradation in mammalian cell cultures, a process referred to as RNA interference (RNAi). In the present study, the potential of RNAi was compared to the traditional antisense approach, acting mainly via RnaseH, for targeting the recombinant rat pain-related cation-channel P2X3 expressed in CHO-K1 and a rat brain tumour-derived cell line, 33B. Downregulation of the P2X3 receptor was evaluated at the mRNA, protein, and functional levels. In this study, four siRNA duplexes induced up to 95% sequence-specific inhibition of the P2X3 mRNA, independent of the type of 2 nt 3'-overhang modification and the location of the targeted sequences. Furthermore, we detected and characterised an independent combinatorial effect of antisense oligonucleotides (ASOs) and RNAi-mediated specific inhibition of the P2X3 receptor. Enhanced downregulation was observed only when siRNA was combined with nonhomologous ASO, targeting distant regions on the common P2X3 mRNA. The two reagents resulted in more efficient downregulation of P2X3 mRNA when administered in combination rather than separately. To our knowledge, this is the first investigation at the molecular level of the potential benefits of mixed antisense and RNAi-mediated treatment for inhibiting expression of a medically relevant pain-related gene.
