Abstract
Small interfering RNAs (siRNAs) have potential to silence virtually any disease-causing gene but require chemical modifications for delivery to the tissue and cell of interest. Previously, we demonstrated that asymmetric, phosphorothioate (PS)-modified, chemically stabilized, cholesterol-conjugated siRNAs, called hsiRNAs, support rapid cellular uptake and efficient mRNA silencing both in cultured cells and in vivo. Here, we systematically evaluated the impact of number, structure, and sequence context of PS-modified backbones on cellular uptake and RNAi-mediated silencing efficacy. We find that PS enhances cellular internalization in a sequence-dependent manner but only when present in a single-stranded but not double-stranded region. Furthermore, the observed increase in cellular internalization did not correlate with functional silencing improvement, indicating that PS-mediated uptake may drive compounds to non-productive sinks. Thus, the primary contributing factor of PS modifications to functional efficacy is likely stabilization rather than enhanced cellular uptake. A better understanding of the relative impact of different chemistries on productive versus non-productive uptake will assist in improved design of therapeutic RNAs.
Highlights
Small interfering RNA-based drugs have therapeutic potential to target and silence any disease-causing gene.[1,2,3] naked siRNAs are prone to degradation by nucleases as well as too large and too hydrophilic to be internalized into cells on their own
Structures and Chemical Modification Patterns of Self-Delivering siRNA Compounds Used in the Study In order to evaluate the extent to which the structural context of PS modifications affects the intracellular trafficking of hydrophobically modified siRNAs, we synthesized a panel of variants of a wellstudied hydrophobic siRNA configuration called hsiRNA.[19] hsiRNAs are asymmetric (20-nt antisense strand, 15-nt sense strand) siRNAs where all sugars are modified with an alternating 20-fluoro (F)/20-O-methyl (OMe) pattern, terminal backbones are protected with PS, and the sense strand is modified with cholesterol
The observed co-localization with endosome antigen 1 (EEA1) and lysosomal associated membrane protein 1 (LAMP1; lysosomal marker) shows very different spatial distribution patterns. hsiRNAs seem to be surrounded within EEA1-enriched endosomes, whereas with LAMP1, Decreasing the Overall PS Content Decreases Cellular Uptake and Silencing Activity of Hydrophobically Modified siRNAs Using this panel of hsiRNAs (Figure 1), we evaluated the impact of oligonucleotide structure and PS content on cellular uptake and silencing activity
Summary
Small interfering RNA (siRNA)-based drugs have therapeutic potential to target and silence any disease-causing gene.[1,2,3] naked siRNAs are prone to degradation by nucleases as well as too large and too hydrophilic to be internalized into cells on their own. A broad range of conjugates can be used to enhance cellular internalization, including N-acetylgalactosamine (GalNAc),[6] cholesterol,[7,8] and docosahexaenoic acid (DHA),[9] which allow the compounds to be internalized through a subset of the endocytic pathway, accumulating to a significant extent in endosomes and lysosomes.[10,11,12] Endosomal entrapment is believed to create an intracellular depot of oligonucleotides, and subsequent slow cytoplasmic release results in continuous loading of the endogenous RNAi enzymatic machinery, the RNA-induced silencing complex (RISC), translating into multi-month efficacy This model relies on the stability of the oligonucleotides inside the highly aggressive biological environment, such as lysosomes, and requires extensive chemical stabilization
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