RESUMEN
The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4⺠T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission.
Asunto(s)
Aptámeros de Nucleótidos/uso terapéutico , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Cuello del Útero/efectos de los fármacos , Genes gag , Genes vif , Infecciones por VIH/prevención & control , Macrófagos/efectos de los fármacos , ARN Interferente Pequeño/uso terapéutico , Receptores CCR5/genética , Quimera por Trasplante/virología , Vagina/efectos de los fármacos , Administración Intravaginal , Animales , Aptámeros de Nucleótidos/administración & dosificación , Secuencia de Bases , Antígenos CD4/genética , Linfocitos T CD4-Positivos/inmunología , Polaridad Celular , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Cuello del Útero/virología , Evaluación Preclínica de Medicamentos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Infecciones por VIH/transmisión , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Datos de Secuencia Molecular , Técnicas de Cultivo de Órganos , ARN Interferente Pequeño/administración & dosificación , Especificidad de la Especie , Quimera por Trasplante/inmunología , Vagina/virologíaRESUMEN
The chemokine receptor CXCR3 is a G protein-coupled receptor found predominantly on T cells that is activated by three ligands as follows: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Previously, we have found that of the three ligands, CXCL11 is the most potent inducer of CXCR3 internalization and is the physiologic inducer of CXCR3 internalization after T cell contact with activated endothelial cells. We have therefore hypothesized that these three ligands transduce different signals to CXCR3. In light of this hypothesis, we sought to determine whether regions of CXCR3 are differentially required for CXCL9, CXCL10, and CXCL11 function. Here we identified two distinct domains that contributed to CXCR3 internalization. The carboxyl-terminal domain and beta-arrestin1 were predominantly required by CXCL9 and CXCL10, and the third intracellular loop was predominantly required by CXCL11. Chemotaxis and calcium mobilization induced by all three CXCR3 ligands were dependent on the CXCR3 carboxyl terminus and the DRY sequence in the third trans-membrane domain. Our findings demonstrate that distinct domains of CXCR3 mediate its functions and suggest that the differential requirement of these domains contributes to the complexity of the chemokine system.