RESUMEN

Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes without inducing chronic toxicity. However, vector therapeutic index is narrow because of a toxic acute response with potentially lethal consequences elicited by high vector doses. Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) are major barriers to efficient hepatocyte transduction. We investigated two small peptides (PP1 and PP2) developed by phage display to block scavenger receptor type A (SR-A) and scavenger receptor expressed on endothelial cells type I (SREC-I), respectively, for enhancement of HDAd-mediated hepatocyte transduction efficiency. Pre-incubation of J774A.1 macrophages with either PP1 or PP2 prior to HDAd infection significantly reduced viral vector uptake. In vivo, fluorochrome-conjugated PP1 and PP2 injected intravenously into mice co-localized with both CD68 and CD31 on KCs and LSECs, respectively. Compared with saline pre-treated animals, intravenous injections of both peptides prior to the injection of an HDAd resulted in up to 3.7- and 2.9-fold increase of hepatic transgene expression with PP1 and PP2, respectively. In addition to greater hepatocyte transduction, compared with control saline injected mice, pre-treatment with either peptide resulted in no increased levels of serum interleukin-6, the major marker of adenoviral vector acute toxicity. In summary, we developed small peptides that significantly increase hepatocyte transduction efficacy and improve HDAd therapeutic index with potential for clinical applications.

Asunto(s)

Adenoviridae/genética , Hepatocitos/metabolismo , Péptidos/farmacología , Receptores Depuradores de Clase A/antagonistas & inhibidores , Receptores Depuradores de Clase F/antagonistas & inhibidores , Transducción Genética , Secuencia de Aminoácidos , Animales , Vectores Genéticos , Virus Helper/genética , Hepatocitos/efectos de los fármacos , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Biblioteca de Péptidos , Péptidos/genética

Asunto(s)

Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Desarrollo Embrionario , Corazón/embriología , Corazón/crecimiento & desarrollo , Homeostasis , Homocigoto , Ratones , Ratones Noqueados , Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/deficiencia

RESUMEN

The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology in adulthood. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). We studied the role of TSHTSHR signaling on gland morphogenesis and differentiation in the mouse embryo using mouse lines deprived either of TSH (pit(dw)pit(dw)) or of a functional TSHR (tshr(hyt)tshr(hyt) and TSHR-knockout lines). The results reported here show that in the absence of either TSH or a functional TSHR, the thyroid gland develops to a normal size, whereas the expression of thyroperoxidase and the sodium/iodide symporter are reduced greatly. Conversely, no relevant changes are detected in the amounts of thyroglobulin and the thyroid-enriched transcription factors TTF-1, TTF-2, and Pax8. These data suggest that the major role of the TSH/TSHR pathway is in controlling genes involved in iodide metabolism such as sodium/iodide symporter and thyroperoxidase. Furthermore, our data indicate that in embryonic life TSH does not play an equivalent role in controlling gland growth as in the adult thyroid.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica/fisiología , Receptores de Tirotropina/fisiología , Transducción de Señal/fisiología , Glándula Tiroides/embriología , Tirotropina/fisiología , Animales , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Diferenciación Celular , Cruzamientos Genéticos , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead , Edad Gestacional , Humanos , Hipotiroidismo/embriología , Hipotiroidismo/genética , Yoduro Peroxidasa/biosíntesis , Yoduro Peroxidasa/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Morfogénesis , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Tamaño de los Órganos , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Tirotropina/deficiencia , Receptores de Tirotropina/genética , Proteínas Recombinantes de Fusión/fisiología , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Simportadores/biosíntesis , Simportadores/genética , Tiroglobulina/biosíntesis , Tiroglobulina/genética , Glándula Tiroides/crecimiento & desarrollo , Glándula Tiroides/patología , Factor Nuclear Tiroideo 1 , Tirotropina/deficiencia , Tirotropina/genética , Transactivadores/biosíntesis , Transactivadores/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética