Abstract
Small interfering RNAs (siRNAs) are a new class of promising therapeutic molecules that can be used for sequence-specific downregulation of disease-causing genes. However, endosomal entrapment of siRNA is a key hurdle for most delivery strategies, limiting the therapeutic effect. Here, we use live-cell microscopy and cytosolic galectin-9 as a sensor of membrane damage, to probe fundamental properties of endosomal escape of cholesterol-conjugated siRNA induced by endosome-disrupting compounds. We demonstrate efficient release of ligand-conjugated siRNA from vesicles damaged by small molecules, enhancing target knockdown up to ∼47-fold in tumor cells. Still, mismatch between siRNA-containing and drug-targeted endolysosomal compartments limits siRNA activity improvement. We also show widespread endosomal damage in macroscopic tumor spheroids after small molecule treatment, substantially improving siRNA delivery and knockdown throughout the spheroid. We believe the strategy to characterize endosomal escape presented here will be widely applicable, facilitating efforts to improve delivery of siRNA and other nucleic acid-based therapeutics.
Highlights
Small interfering RNAs are a new class of promising therapeutic molecules that can be used for sequence-specific downregulation of disease-causing genes
Consistent with previous reports[28], staining HeLa cells with anti-galectin-3 after 24 h incubation with amitriptyline, siramesine, or chloroquine revealed the formation of intracellular galectin foci, as evidence of membrane damages (Fig. 1a)
We demonstrate the applicability of galectin-9 as a highly sensitive membrane damage sensor, to directly visualize and measure properties of endosomal escape of ligand-conjugated Small interfering RNAs (siRNA)
Summary
Small interfering RNAs (siRNAs) are a new class of promising therapeutic molecules that can be used for sequence-specific downregulation of disease-causing genes. We demonstrate efficient release of ligand-conjugated siRNA from vesicles damaged by small molecules, enhancing target knockdown up to ∼47-fold in tumor cells. We show widespread endosomal damage in macroscopic tumor spheroids after small molecule treatment, substantially improving siRNA delivery and knockdown throughout the spheroid. Electron microscopy studies of liver tissues suggested that LNPs mediate siRNA release in the early stages of the endosomal system[8]. The vast majority of the internalized molecules are sequestered in the endosomal system, unable to reach the cytosol and the RNA interference (RNAi) machinery[12]. This can be partially overcome by targeting highly abundant cell surface receptors. No additional ligand–receptor pair of equal potency has been found, and ASGPR expression is largely restricted to the liver
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