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GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells

ABSTRACT: GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells

PROVIDER: PRJNA830477 | ENA |

REPOSITORIES: ENA

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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.

2022-04-26 | GSE201237 | GEO

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

| PRJNA723544 | ENA

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

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

2021-04-22 | GSE172485 | GEO

Antisense oligonucleotide activity in tumour cells is influenced by intracellular LBPA distribution and 6 extracellular vesicles recycling

Project description: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. We compared intracellular traffic of anti KRAS antisense oligonucleotide (AZD4785) in good and poor productive uptake tumour cells, PC9 and LK2 respectively. We found 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 cells recycle AZD4785 in the extracellular vesicles (EVs) however AZD4785 quantification by advanced mass spectrometry and proteomic analysis revealed that LK2 recycles more AZD4785 and RNA-binding proteins. Finally, stimulating LBPA intracellular production or blocking EV recycling enhanced AZD4785 activity in LK2 but not in PC9 cells thus offering novel strategy to possibly enhance ASO potency in poor productive uptake tumour cells.

2021-11-10 | PXD027804 | Pride

Liver proteome upon in vivo N-acetylgalactosamine-conjugated antisense oligonucleotide silencing of ceramide synthase 2

Project description:Mouse liver proteome was investigated upon in vivo mouse treatment with a N-acetylgalactosamine-conjugated antisense oligonucleotide engineered to silence ceramide synthase 2 specifically in hepatocytes in vivo. The data is a part of a study on the involvement of ceramide enzymatic machinery in cardiovasular disorders and its potential as a target for the disease treatment.

2023-05-10 | PXD025957 | Pride

Expression data from dosing an antisense oligonucleotide in mice I

Project description:We used microarrays to globally profile the gene expression changes observed in liver after 3 days when dosing an antisense oligonucleotide in mice

2018-04-30 | GSE100697 | GEO

Expression data from transfection of an antisense oligonucleotide in 518A2 cells

Project description:We used microarrays to globally profile the gene expression changes observed after 3 days when transfecting an antisense oligonucleotide in 518A2 cells

2017-04-21 | GSE93185 | GEO

Expression data from dosing an antisense oligonucleotide in mice

Project description:Expression data from dosing an antisense oligonucleotide in mice

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Transcriptional analysis of select CfMNPV genes and some antisense transcripts by an oligonucleotide microarray

Project description:The temporal expression of the 23 CfMNPV genes representing all four temporal classes including its 7 unique genes were determined by a modified oligonucleotide-based two-channel DNA microarray. Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene was also detected by the array analysis. The expression of four host genes varied several fold throughout virus infection. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements. We first developed a novel normalization protocol using Cy5-labeled CfMNPV viral genomic DNA (vgDNA) as equimolar reference standards for each probe in order to overcome the inherent variability problem of the traditional microarray normalization procedures including use of internal standards. Cy3-labeled cDNA was from total RNA isolated at different times post infection of Cf203 insect cells infected with CfMNPV. Host genes were unsuitable for normalization between microarrays. The DNA microarray results were selectively validated by quantitative RT-PCR (qRT-PCR). The nature of the polyhedrin antisense transcription was further investigated using long range RT-PCR analysis. Keywords: Time course, detection of antisense transcripts, viral genomic DNA normalization

2007-05-01 | GSE4783 | GEO

MaTAR25 RNA Antisense Pulldown and Mass Spectrometry

Project description:It has been suggested that lncRNAs can interact with transcriptional regulators/co-factors to form ribonucleoprotein (RNP) complexes to regulate the expression of downstream genes in the cell nucleus. To identify potential interacting proteins of MaTAR25, we used two different paired sets of biotin-labeled antisense oligonucleotides targeting MaTAR25 for native RNA antisense oligonucleotide pull-down (RAP) in 4T1 cells followed by qRT-PCR to assess pull-down efficiency. Samples were also eluted from beads for mass spectrometry isobaric tags for relative and absolute quantitative (MS-iTRAQ) analysis to identify proteins that bind to MaTAR25, and PPIB as the corresponding control. We ranked the candidate interactors based on detectable peptides above background in both pair sets of oligonucleotide pull-downs, and selected candidates with at least 2-fold enrichment compared to corresponding PPIB oligo pull-down.

2020-12-23 | PXD017398 | Pride

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