Differential uptake, kinetics and mechanisms of intracellular trafficking of next-generation antisense oligonucleotides across human cancer cell lines

Abstract

Antisense oligonucleotides (ASOs) modulate cellular target gene expression through direct binding to complementary RNA. Advances in ASO chemistry have led to the development of phosphorothioate (PS) ASOs with constrained-ethyl modifications (cEt). These next-generation cEt-ASOs can enter cells without transfection reagents. Factors involved in intracellular uptake and trafficking of cEt-ASOs leading to successful target knockdown are highly complex and not yet fully understood. AZD4785 is a potent and selective therapeutic KRAS cEt-ASO currently under clinical development for the treatment of cancer. Therefore, we used this to investigate mechanisms of cEt-ASO trafficking across a panel of cancer cells. We found that the extent of ASO-mediated KRAS mRNA knockdown varied significantly between cells and that this did not correlate with bulk levels of intracellular accumulation. We showed that in cells with good productive uptake, distribution of ASO was perinuclear and in those with poor productive uptake distribution was peripheral. Furthermore, ASO rapidly trafficked to the late endosome/lysosome in poor productive uptake cells compared to those with more robust knockdown. An siRNA screen identified several factors mechanistically involved in productive ASO uptake, including the endosomal GTPase Rab5C. This work provides novel insights into the trafficking of cEt-ASOs and mechanisms that may determine their cellular fate.

A protein tertiary structure mimetic modulator of the Hippo signalling pathway

“…3b) highlighting the protective effect that results from macrocyclization. To assess the cell penetration properties of unlabelled analogues, we determined their cytoplasmic and nuclear concentrations using a mass spectrometry-based methodology [25][26][27] and the Tat peptide as reference. RKO cells were incubated (c(peptide) = 25 µM) for 90 min and 24 h, then the cells were lysed and fractionated to isolate cytoplasmic and nuclear contents for subsequent UPLC-MS analysis.…”

A critical analysis of methods used to investigate the cellular uptake and subcellular localization of RNA therapeutics

“…The fluorescence from the secondary antibody is detected by fluorescence microscopy, and fluorescence can be quantitated to measure the relative amount of internalized oligonucleotide. Additional co-localization studies with membrane proteins involved in endocytosis can aid in the study of subcellular localization ( 44 , 105 , 107 ). Immunofluorescence assays can be performed using unlabeled oligonucleotides, and the use of antibodies for detection renders this method highly specific.…”

“…While NMR-based quantitation suffers from a lack of sensitivity, mass spectrometry methods are notable for their excellent sensitivity. Mass spectrometry methods are becoming more and more common for measuring the total cellular uptake of RNA therapeutics, both in cultured cells and ex vivo ( 42 , 107 , 116–119 ). Cells or tissues are homogenized and lysed, and the cleared lysates are analyzed by liquid chromatography and mass spectrometry (LC–MS, Figure 4C ) ( 42 , 107 , 116–119 ).…”

Transcription and Translation Inhibitors in Cancer Treatment

“…AZD4785 was previously shown to efficiently deplete KRAS and was associated with an antitumor effect in mice (Ross et al, 2017;Sacco et al, 2019). In addition, cellular trafficking and localization of AZD4785 across different tumor cell lines have been characterized and was found to vary (Linnane et al, 2019). Following completion of a phase I trial the molecule was demonstrated to be safe and well-tolerated, but it was discontinued by AstraZeneca because of its insufficient efficacy possibly due to targeting both mutant and wild-type KRAS mRNA (Yang et al, 2019).…”