RESUMEN
One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc-siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc-siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.
Asunto(s)
Acetilgalactosamina/metabolismo , Receptor de Asialoglicoproteína/metabolismo , Portadores de Fármacos , Silenciador del Gen , Prealbúmina/genética , ARN Interferente Pequeño/metabolismo , Acetilgalactosamina/química , Animales , Proteínas Argonautas/genética , Receptor de Asialoglicoproteína/genética , Transporte Biológico , Estabilidad de Medicamentos , Femenino , Glicoconjugados/química , Glicoconjugados/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , Nanopartículas/metabolismo , Prealbúmina/antagonistas & inhibidores , Prealbúmina/metabolismo , ARN Interferente Pequeño/genética , Factores de TiempoRESUMEN
The 5'-monophosphate group plays an important role in strand selection during gene silencing mediated by small-interfering RNA. We show that blocking of 5' phosphorylation of the sense strand by introducing a 5'-morpholino modification improves antisense strand selection and RNAi activity. The 5'-morpholino modification of the antisense strand triggers complete loss of activity.
Asunto(s)
Morfolinos/química , ARN Interferente Pequeño/química , Animales , Apolipoproteína B-100 , Apolipoproteínas B/genética , Proteínas Argonautas/genética , Silenciador del Gen , Humanos , Ratones , Modelos Moleculares , Morfolinos/síntesis química , Morfolinos/genética , Interferencia de ARN , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/genéticaRESUMEN
5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5'-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5'-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5'-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs. In this study, we report the synthesis of 5'-C-malonyl RNA, a 5'-monophosphate bioisostere. A 5'-C-malonyl-modified nucleotide was incorporated at the 5'-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. In vitro silencing activity, in vitro metabolic stability, and in vitro RISC loading of 5'-C-malonyl siRNA was compared to corresponding 5'-phosphorylated and 5'-nonphosphorylated siRNAs. The 5'-C-malonyl siRNAs showed sustained or improved in vitro gene silencing and high levels of Ago2 loading and conferred dramatically improved metabolic stability to the antisense strand of the siRNA duplexes. In silico modeling studies indicate a favorable fit of the 5'-C-malonyl group within the 5'-phosphate binding pocket of human Ago2MID domain.
Asunto(s)
Silenciador del Gen , Malonatos/química , ARN Interferente Pequeño/genética , Animales , Células Cultivadas , Ratones , FosforilaciónRESUMEN
The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.