RESUMO
siRNAs confer sequence specific and robust silencing of mRNA. By virtue of these properties, siRNAs have become therapeutic candidates for disease intervention. However, their use as therapeutic agents can be hampered by unintended off-target effects by either or both strands of the siRNA duplex. We report here that unlocked nucleobase analogs (UNAs) confer desirable properties to siRNAs. Addition of a single UNA at the 5'-terminus of the passenger strand blocks participation of the passenger strand in RISC-mediated target down-regulation with a concomitant increase in guide strand activity. Placement of a UNA in the seed region of the guide strand prevents miRNA-like off-target silencing without compromising siRNA activity. Most significantly, combined substitution of UNA at the 3'-termini of both strands, the addition of a UNA at the 5'-terminus of the passenger strand, and a single UNA in the seed region of the guide strand, reduced the global off-target events by more than 10-fold compared to unmodified siRNA. The reduction in off-target events was specific to UNA placement in the siRNA, with no apparent new off-target events. Taken together, these results indicate that when strategically placed, UNA substitutions have important implications for the design of safe and effective siRNA-based therapeutics.
Assuntos
Interferência de RNA , RNA Interferente Pequeno/química , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , MicroRNAs/metabolismoRESUMO
Harnessing RNA interference (RNAi) to silence aberrant gene expression is an emerging approach in cancer therapy. Selective inhibition of an overexpressed gene via RNAi requires a highly efficacious, target-specific short interfering RNA (siRNA) and a safe and efficient delivery system. We have developed siRNA constructs (UsiRNA) that contain unlocked nucleobase analogs (UNA) targeting survivin and polo-like kinase-1 (PLK1) genes. UsiRNAs were encapsulated into dialkylated amino acid-based liposomes (DiLA(2)) containing a nor-arginine head group, cholesteryl hemisuccinate (CHEMS), cholesterol and 1, 2-dimyristoyl-phosphatidylethanolamine-polyethyleneglycol 2000 (DMPE-PEG2000). In an orthotopic bladder cancer mouse model, intravesical treatment with survivin or PLK1 UsiRNA in DiLA(2) liposomes at 1.0 and 0.5 mg/kg resulted in 90% and 70% inhibition of survivin or PLK1 mRNA, respectively. This correlated with a dose-dependent decrease in tumor volumes which was sustained over a 3-week period. Silencing of survivin and PLK1 mRNA was confirmed to be RNA-induced silencing complex mediated as specific cleavage products were detected in bladder tumors over the duration of the study. This report suggests that intravesical instillation of survivin or PLK1 UsiRNA can serve as a potential therapeutic modality for treatment of bladder cancer.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Inibidoras de Apoptose/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/terapia , Administração Intravesical , Animais , Western Blotting , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Colesterol/administração & dosagem , Ésteres do Colesterol/administração & dosagem , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Lipossomos/administração & dosagem , Camundongos , Camundongos Nus , Fosfatidiletanolaminas/administração & dosagem , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , Survivina , Neoplasias da Bexiga Urinária/patologia , Quinase 1 Polo-LikeRESUMO
RNA interference (RNAi) involves sequence-specific downregulation of target genes, leading to gene silencing in vitro and in vivo. Synthetic small interfering RNAs (siRNAs), formulated with appropriate delivery agents, can serve as effective tools for RNAi-based therapeutics. The potential of siRNA to provide antiviral activity has been studied extensively in many respiratory viruses, including influenza virus, wherein specific siRNAs target highly-conserved regions of influenza viral genome, leading to potent inhibition of viral RNA replication. Despite various delivery strategies, such as polycations and liposomes that have been employed to formulate siRNAs, effective delivery modalities are still needed. Although current strategies can provide significant biodistribution and delivery into lungs allowing gene silencing, complete protection and prolonged survival rates against multiple strains of influenza virus still remains a key challenge. Here, we describe methods and procedures pertaining to screening and selection of highly effective influenza-specific siRNAs in cell culture, in mice, and in the ferret model. This will be potentially useful to evaluate RNAi as a therapeutic modality for future clinical application.