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KRAS and MEK activation promote antisense oligonucleotide uptake and activity by limiting collagen production

ABSTRACT: Using RNAseq we describe transcriptional changes downstream of MEK1 affecting RNA kncokdown following free uptake RNaseH1 gapmer antisense oligonucleotides

ORGANISM(S): Homo sapiens

PROVIDER: GSE172485 | GEO | 2021/04/22

REPOSITORIES: GEO

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KRAS and MEK activation promote antisense oligonucleotide uptake and activity by limiting collagen production

Project description:KRAS and MEK activation promote antisense oligonucleotide uptake and activity by limiting collagen production

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GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells

Project description:Antisense oligonucleotides (ASOs) are short synthetic nucleic acids that recognize and bind to complementary RNA to modulate gene expression. It is well-established that single-stranded, phosphorothioate modified ASOs enter cells independent of carrier molecules, primarily via endocytic pathways, but that only a small portion of internalized ASO is released into the cytosol and/or nucleus rendering the majority of ASO as inaccessible to the targeted RNA. Identifying pathways that can increase the available ASO pool is valuable as a research tool and therapeutically. Here, we conducted a functional genomic screen for ASO activity by engineering GFP splice reporter cells and applying genome wide CRISPR gene activation. The screen can identify factors that enhance ASO splice modulation activity. Characterization of hit genes uncovered GOLGA8, a largely uncharacterized protein, as a novel positive regulator enhancing ASO activity by ~2-fold. Bulk ASO uptake is 5-fold higher in GOLGA8 overexpressing cells where GOLGA8 and ASOs are observed in the same intracellular compartments. We find GOLGA8 is highly localized to the trans-Golgi and readily detectable at the plasma membrane. Interestingly, overexpression of GOLGA8 increased activity for both splice modulation and RNase H1-dependent ASOs. Taken together, these results support a novel role for GOLGA8 in productive ASO uptake.

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RNA-seq analysis of HeLa cells treated with GapmeR antisense oligonucleotides

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Silencing of NFYC-AS1 by Gapmer antisense oligonucleotides and CRISPR/Cas9 TSS deletion.

Project description:NFYC-AS1 is an overlapping antisense RNA transcribed head-to-head to NFYC sense gene, encoding for the subunit C of NF-Y transcription factor, which is known as master regulator of cell cycle and proliferation in normal and tumor cells. Here we performed NFYC-AS1 silencing in lung squamous carcinoma H520 cells by Gapmer antisense oligonucleotides and CRISPR/Cas9 TSS deletion. Afterwards, we performed differentially expressed analysis and gene set enrichement analysis to investigate on NFYC-AS1 function and mechanism of action.

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Silencing of NFYC-AS1 by Gapmer antisense oligonucleotides and CRISPR/Cas9 TSS deletion.

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In vivo uptake of antisense oligonucleotide drugs predicted by ab initio quantum mechanical calculations.

Project description:Liver and kidney uptake and antisense activity is studied for a series of Locked Nucleic Acid (LNA) oligonucleotides with fully stereo-defined, internucleoside linkages. These stereo-specific phosphorothioates are made with a newly developed synthesis method and are being analyzed both theoretically and experimentally. Their structures are obtained theoretically by using many-body Schrödinger equations applied to a group of 11 stereo-defined LNA antisense oligonucleotides selected for biological experiments. The fully converged electronic structures were obtained from ab initio quantum calculations providing the specific electronic structures. One important result was the observation that the calculated electronic structure, represented by the iso-surface area of the electron density in Å2, correlated linearly with LNA oligonucleotide uptake in the liver and kidney. This study also shows that more complex biological phenomena, such as drug activity, will require more molecular and cellular identifiers than used here before a correlation can be found. Establishing biological correlations between quantum mechanical (QM) calculated structures and antisense oligonucleotides is novel, and this method may constitute new tools in drug discovery.

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Scavenger Receptor Class A1 Mediates Uptake of Morpholino Antisense Oligonucleotide into Dystrophic Skeletal Muscle.

Project description:Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) is a promising treatment strategy for Duchenne muscular dystrophy (DMD). The most significant limitation of these clinically used compounds is their lack of delivery systems that target muscles; thus, cell-penetrating peptides are being developed to enhance uptake into muscles. Recently, we reported that uptake of peptide-conjugated PMOs into myofibers was mediated by scavenger receptor class A (SR-A), which binds negatively charged ligands. However, the mechanism by which the naked PMOs are taken up into fibers is poorly understood. In this study, we found that PMO uptake and exon-skipping efficiency were promoted in dystrophin-deficient myotubes via endocytosis through a caveolin-dependent pathway. Interestingly, SR-A1 was upregulated and localized in juxtaposition with caveolin-3 in these myotubes and promoted PMO-induced exon skipping. SR-A1 was also upregulated in the skeletal muscle of mdx52 mice and mediated PMO uptake. In addition, PMOs with neutral backbones had negative zeta potentials owing to their nucleobase compositions and interacted with SR-A1. In conclusion, PMOs with negative zeta potential were taken up into dystrophin-deficient skeletal muscle by upregulated SR-A1. Therefore, the development of a drug delivery system targeting SR-A1 could lead to highly efficient exon-skipping therapies for DMD.

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Knockdown of circular RNAs using LNA-modified antisense oligonucleotides

Project description:Circular RNAs (circRNAs) constitute an abundant class of covalently closed non-coding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. Here, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We identified 5807 circRNAs in total RNA sequencing data from 4 liver cancer cell lines and used the back splice junction (BSJ) sequences of 7 validated circRNAs as target sites for designing different LNA-modified ASOs for circRNA knockdown. We found that while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript and may also cause degradation of unintended off-targets. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs or LNA/DNA mixmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes or predicted off-targets. In summary, our results demonstrate that LNA-modified ASOs complementary to BSJ sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.

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2018-10-12 | E-MTAB-6529 | biostudies-arrayexpress

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