GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells
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, bu...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-06-01
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| Series: | Molecular Therapy: Nucleic Acids |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253123000768 |
| _version_ | 1797848261320507392 |
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| author | Moira A. McMahon Meghdad Rahdar Swagatam Mukhopadhyay Huynh-Hoa Bui Christopher Hart Sagar Damle Margo Courtney Michael W. Baughn Don W. Cleveland C. Frank Bennett |
| author_facet | Moira A. McMahon Meghdad Rahdar Swagatam Mukhopadhyay Huynh-Hoa Bui Christopher Hart Sagar Damle Margo Courtney Michael W. Baughn Don W. Cleveland C. Frank Bennett |
| author_sort | Moira A. McMahon |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-09T18:24:37Z |
| format | Article |
| id | doaj.art-b4603aa807a3456aa6ae0c476b922870 |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-04-09T18:24:37Z |
| publishDate | 2023-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-b4603aa807a3456aa6ae0c476b9228702023-04-12T04:11:42ZengElsevierMolecular Therapy: Nucleic Acids2162-25312023-06-0132289301GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cellsMoira A. McMahon0Meghdad Rahdar1Swagatam Mukhopadhyay2Huynh-Hoa Bui3Christopher Hart4Sagar Damle5Margo Courtney6Michael W. Baughn7Don W. Cleveland8C. Frank Bennett9Ionis Pharmaceuticals, Inc., Carlsbad, CA 92010, USA; Corresponding author: Moira A. McMahon, Ionis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, CA 92010, USA.Ionis Pharmaceuticals, Inc., Carlsbad, CA 92010, USAIonis Pharmaceuticals, Inc., Carlsbad, CA 92010, USAIonis Pharmaceuticals, Inc., Carlsbad, CA 92010, USAIonis Pharmaceuticals, Inc., Carlsbad, CA 92010, USAIonis Pharmaceuticals, Inc., Carlsbad, CA 92010, USAIonis Pharmaceuticals, Inc., Carlsbad, CA 92010, USALudwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA 92093-0670, USALudwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA 92093-0670, USAIonis Pharmaceuticals, Inc., Carlsbad, CA 92010, USAAntisense 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.http://www.sciencedirect.com/science/article/pii/S2162253123000768MT: Oligonucleotides: Therapies and Applicationsantisense oligonucleotideASOCRISPR-Casgene activationGOLGA8 |
| spellingShingle | Moira A. McMahon Meghdad Rahdar Swagatam Mukhopadhyay Huynh-Hoa Bui Christopher Hart Sagar Damle Margo Courtney Michael W. Baughn Don W. Cleveland C. Frank Bennett GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells Molecular Therapy: Nucleic Acids MT: Oligonucleotides: Therapies and Applications antisense oligonucleotide ASO CRISPR-Cas gene activation GOLGA8 |
| title | GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells |
| title_full | GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells |
| title_fullStr | GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells |
| title_full_unstemmed | GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells |
| title_short | GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells |
| title_sort | golga8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells |
| topic | MT: Oligonucleotides: Therapies and Applications antisense oligonucleotide ASO CRISPR-Cas gene activation GOLGA8 |
| url | http://www.sciencedirect.com/science/article/pii/S2162253123000768 |
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