Differential uptake, kinetics and mechanisms of intracellular trafficking of next-generation antisense oligonucleotides across human cancer cell lines.
Endosomes
/ genetics
Gene Expression Regulation, Neoplastic
/ drug effects
Gene Knockdown Techniques
HT29 Cells
Humans
Neoplasms
/ genetics
Oligonucleotides, Antisense
/ chemistry
Phosphorothioate Oligonucleotides
/ chemistry
Proto-Oncogene Proteins p21(ras)
/ genetics
RNA, Messenger
/ genetics
RNA, Small Interfering
/ genetics
rab5 GTP-Binding Proteins
/ genetics
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
21 05 2019
21 05 2019
Historique:
accepted:
18
03
2019
revised:
11
03
2019
received:
20
12
2018
pubmed:
31
3
2019
medline:
22
11
2019
entrez:
31
3
2019
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 30927008
pii: 5423608
doi: 10.1093/nar/gkz214
pmc: PMC6511877
doi:
Substances chimiques
KRAS protein, human
0
Oligonucleotides, Antisense
0
Phosphorothioate Oligonucleotides
0
RNA, Messenger
0
RNA, Small Interfering
0
RAB5C protein, human
EC 3.6.1.-
Proto-Oncogene Proteins p21(ras)
EC 3.6.5.2
rab5 GTP-Binding Proteins
EC 3.6.5.2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
4375-4392Subventions
Organisme : Medical Research Council
ID : MR/P01058X/1
Pays : United Kingdom
Informations de copyright
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
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