RESUMEN
Small interfering RNAs (siRNAs) induce robust degradation of homologous mRNAs. Highly specific silencing of target genes makes siRNA an interesting tool in drug development. However, several non-specific effects complicate the use of RNA interference (RNAi). One of the most prevalent unspecific effects is triggering the innate immune system in mammals. In parallel, activating the immune system may open the possibility to develop dual siRNAs for treatment of a variety of diseases including cancer. Here, we show that the best use of unmodified siRNAs for RNAi and immune activation depends on the delivery system, formulation condition, sequence and siRNA design concerning ORN motifs. Testing several commercial delivery systems identified that the optimal siRNAs for dual functions should contain TLR7/8 ORN motifs at least in the antisense strand and be delivered by either Dharmafect or HiPerfect. Superfect delivery system only activates TLR7 and opens new capabilities in RNAi and immune activation.
Asunto(s)
Leucocitos Mononucleares/metabolismo , Motivos de Nucleótidos , ARN Interferente Pequeño/genética , Receptor Toll-Like 7/genética , Receptor Toll-Like 8/genética , Secuencia de Bases , Línea Celular , Citocinas/metabolismo , Vectores Genéticos , Humanos , Inmunidad Innata/genética , Inmunización/métodos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Datos de Secuencia Molecular , FN-kappa B/genética , FN-kappa B/metabolismo , Motivos de Nucleótidos/genética , Interferencia de ARNRESUMEN
The toll-like receptors (TLRs) 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. Certain GU- or AU-rich RNA sequences were described to differentiate between human TLR7- and TLR8-mediated immune effects. Those single-stranded RNA molecules require endosomal delivery for stabilization against ribonucleases. We have discovered RNA sequences that preferentially activate TLR7, form higher ordered structures, and do not require specific cellular delivery. In addition, a dual activation of TLR8 and TLR9 without affecting TLR7 can be achieved by chimeric molecules consisting of GU-rich RNA and Cytosin (C) phosphordiester or phosphorthioat (p) guanine (CpG) motif DNA sequences. Such chimeras stimulate TLR9-mediated type I interferon (IFN) and TLR8-depending proinflammatory cytokine and chemokine production upon primary human cell activation. However, an RNA-dependent TLR7 IFN-α cytokine release is suppressed by the phosphorothioate DNA sequence contained in the chimeric molecule. To convert the immune response of a single-stranded RNA from TLR7/8 to TLR9, a simple chemical modification at the 5' end proves to be sufficient. Such 8-oxo-2'-deoxy-guanosine or 8-bromo-2'-deoxy-guanosine modifications of the first guanosine in GU-rich single-stranded RNAs convert the immune response to include TLR9 activation and demonstrate strong additive effects for type I IFN immune responses in human primary cells.