Abstract
Next generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad application. Here we compare intracellular traffic of anti KRAS antisense oligonucleotide (AZD4785) in tumour cell lines PC9 and LK2, with good and poor productive uptake, respectively. We find that the majority of AZD4785 is rapidly delivered to CD63+late endosomes (LE) in both cell lines. Importantly, lysobisphosphatidic acid (LBPA) that triggers ASO LE escape is presented in CD63+LE in PC9 but not in LK2 cells. Moreover, both cell lines recycle AZD4785 in extracellular vesicles (EVs); however, AZD4785 quantification by advanced mass spectrometry and proteomic analysis reveals that LK2 recycles more AZD4785 and RNA-binding proteins. Finally, stimulating LBPA intracellular production or blocking EV recycling enhances AZD4785 activity in LK2 but not in PC9 cells thus offering a possible strategy to enhance ASO potency in tumour cells with poor productive uptake of ASOs.
Highlights
Generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad application
We found that bulk of the internalised AZD4785 is delivered to CD63+late endosomes (LE) and productive uptake is influenced by lysobisphosphatidic acid (LBPA) spatial distribution—with LBPA colocalised with CD63+LE in PC9 cells whilst LBPA presented in LEs distinct from CD63+LE across LK2 cells
To visualise LE’s, we used two LE markers, tetraspanin CD6327 and LBPA, a lipid which is exclusively generated in LE where it enables ASO endosomal escape by mediating back-fusion of ASO-loaded intraluminal vesicles with the LE membrane[15]
Summary
Generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad application. Stimulating LBPA intracellular production or blocking EV recycling enhances AZD4785 activity in LK2 but not in PC9 cells offering a possible strategy to enhance ASO potency in tumour cells with poor productive uptake of ASOs. 1234567890():,; Antisense oligonucleotides (ASOs) are novel, highly specific nucleic acids designed to silence target genes by forming complimentary heteroduplex with target’s mRNA and mediating mRNA degradation[1–3]. Stimulating LBPA production or inhibiting EV recycling pathway by using small molecules enhanced AZD4785 productive uptake in LK2 cells but not in PC9 indicating specificity towards poor productive uptake cell lines Targeting these intracellular pathways could be exploited to boost ASO productive uptake and activity in poor productive tumour cells and, possibly, even across other pathologies
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