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

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

PROVIDER: PRJNA723544 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR14294600.fastq.gz	Fastqsanger.gz
	SRR14294601.fastq.gz	Fastqsanger.gz
	SRR14294602.fastq.gz	Fastqsanger.gz
	SRR14294603.fastq.gz	Fastqsanger.gz
	SRR14294604.fastq.gz	Fastqsanger.gz

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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

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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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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

Limiting Mrs2-Dependent Mg2+ Uptake Induces Metabolic Programming in Prolonged Dietary Stress

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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 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 2- to 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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Project description:C9orf72-linked amyotrophic lateral sclerosis (c9ALS) is driven by an intronic G4C2 repeat expansion, leading to the production of toxic RNA transcripts and dipeptide repeat proteins (DPRs). A clinical trial using the antisense oligonucleotide (ASO), BIIB078, to target these transcripts was discontinued after failing to meet secondary efficacy endpoints, despite a reduction in DPRs in cerebrospinal fluid (CSF). The extent of target engagement in the central nervous system (CNS) and the relevance of pharmacodynamic CSF biomarkers following BIIB078 treatment remain unclear. Post-mortem analysis of CNS tissue from six BIIB078-treated c9ALS patients revealed widespread intracellular BIIB078 distribution in the spinal cord and brain regions, persisting for over a year after the last dose. C9orf72 mRNA levels were inversely correlated with BIIB078 concentration, indicating partial target engagement. Despite reductions in CSF DPRs, these proteins and phosphorylated TDP-43 remained abundant in the brain and spinal cord, suggesting that CSF biomarkers may not fully reflect CNS target engagement. Additionally, sustained increases in inflammation biomarkers, including CCL26, GDF15, and other cytokines, were observed. Global proteomic analysis of spinal cord tissue revealed significant changes in c9ALS cases that were not normalized in BIIIB078 treated cases. Notably, proteome-wide correlations with BIIB078 concentration in the spinal cord identified hydrolytic nucleases, including RNASET2 and DNASE2, to be increased in BIIB078 treated cases. Thus, while BIIB078 demonstrated widespread and sustained distribution and partial target engagement, it did not significantly impact key proteomic and pathological features in the CNS of c9ALS patients. Consequently, ceasing strategies that target the G4C2 sense strand may be premature.

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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.

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Project description:Calmodulinopathies are rare inherited arrhythmia syndromes caused by dominant gain of function variants in one of three genes, CALM1, CALM2, and CALM3, which each encode the identical calmodulin (CaM) protein. We hypothesized that antisense oligonucleotide (ASO)-mediated depletion of an affected calmodulin gene would ameliorate disease manifestations, while the other two calmodulin genes would preserve CaM level and function. Here we tested this hypothesis using human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) and mouse models of CALM1 pathogenic variants. Human CALM1F142L/+ iPSC-CMs exhibited prolonged action potentials, modeling congenital long QT syndrome. CALM1-depleting ASOs did not alter CaM protein level and normalized repolarization of CALM1F142L/+ iPSC-CMs. Similarly, an ASO targeting murine Calm1 depleted Calm1 transcript without affecting CaM protein level. This ASO alleviated drug-induced arrhythmia in CalmN98S/+ mice without causing observable toxicity. These results provide proof-of-concept that ASOs targeting individual calmodulin genes are potentially effective and safe therapies for calmodulinopathies.

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