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RAB18 is a key regulator of GalNAc-conjugated siRNA-induced silencing in Hep3B cells

Small interfering RNA (siRNA) therapeutics have developed rapidly in recent years, despite the challenges associated with delivery of large, highly charged nucleic acids. Delivery of siRNA therapeutics to the liver has been established, with conjugation of siRNA to N-acetylgalactosamine (GalNAc) pro...

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Autores principales: Lu, Jiamiao, Swearingen, Elissa, Hardy, Miki, Collins, Patrick, Wu, Bin, Yuan, Eric, Lu, Daniel, Li, Chi-Ming, Wang, Songli, Ollmann, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9035644/
https://www.ncbi.nlm.nih.gov/pubmed/35505960
http://dx.doi.org/10.1016/j.omtn.2022.04.003
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author Lu, Jiamiao
Swearingen, Elissa
Hardy, Miki
Collins, Patrick
Wu, Bin
Yuan, Eric
Lu, Daniel
Li, Chi-Ming
Wang, Songli
Ollmann, Michael
author_facet Lu, Jiamiao
Swearingen, Elissa
Hardy, Miki
Collins, Patrick
Wu, Bin
Yuan, Eric
Lu, Daniel
Li, Chi-Ming
Wang, Songli
Ollmann, Michael
author_sort Lu, Jiamiao
collection PubMed
description Small interfering RNA (siRNA) therapeutics have developed rapidly in recent years, despite the challenges associated with delivery of large, highly charged nucleic acids. Delivery of siRNA therapeutics to the liver has been established, with conjugation of siRNA to N-acetylgalactosamine (GalNAc) providing durable gene knockdown in hepatocytes following subcutaneous injection. GalNAc binds the asialoglycoprotein receptor (ASGPR) that is highly expressed on hepatocytes and exploits this scavenger receptor to deliver siRNA across the plasma membrane by endocytosis. However, siRNA needs to access the RNA-induced silencing complex (RISC) in the cytoplasm to provide effective gene knockdown, and the entire siRNA delivery process is very inefficient, likely because of steps required for endosomal escape, intracellular trafficking, and stability of siRNA. To reveal the cellular factors limiting delivery of siRNA therapeutics, we performed a genome-wide pooled knockout screen on the basis of delivery of GalNAc-conjugated siRNA targeting the HPRT1 gene in the human hepatocellular carcinoma line Hep3B. Our primary genome-wide pooled knockout screen identified candidate genes that when knocked out significantly enhanced siRNA efficacy in Hep3B cells. Follow-up studies indicate that knockout of RAB18 improved the efficacy of siRNA delivered by GalNAc, cholesterol, or antibodies, but not siRNA delivered by Lipofectamine transfection, suggesting a role for RAB18 in siRNA delivery and intracellular trafficking.
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spelling pubmed-90356442022-05-02 RAB18 is a key regulator of GalNAc-conjugated siRNA-induced silencing in Hep3B cells Lu, Jiamiao Swearingen, Elissa Hardy, Miki Collins, Patrick Wu, Bin Yuan, Eric Lu, Daniel Li, Chi-Ming Wang, Songli Ollmann, Michael Mol Ther Nucleic Acids Original Article Small interfering RNA (siRNA) therapeutics have developed rapidly in recent years, despite the challenges associated with delivery of large, highly charged nucleic acids. Delivery of siRNA therapeutics to the liver has been established, with conjugation of siRNA to N-acetylgalactosamine (GalNAc) providing durable gene knockdown in hepatocytes following subcutaneous injection. GalNAc binds the asialoglycoprotein receptor (ASGPR) that is highly expressed on hepatocytes and exploits this scavenger receptor to deliver siRNA across the plasma membrane by endocytosis. However, siRNA needs to access the RNA-induced silencing complex (RISC) in the cytoplasm to provide effective gene knockdown, and the entire siRNA delivery process is very inefficient, likely because of steps required for endosomal escape, intracellular trafficking, and stability of siRNA. To reveal the cellular factors limiting delivery of siRNA therapeutics, we performed a genome-wide pooled knockout screen on the basis of delivery of GalNAc-conjugated siRNA targeting the HPRT1 gene in the human hepatocellular carcinoma line Hep3B. Our primary genome-wide pooled knockout screen identified candidate genes that when knocked out significantly enhanced siRNA efficacy in Hep3B cells. Follow-up studies indicate that knockout of RAB18 improved the efficacy of siRNA delivered by GalNAc, cholesterol, or antibodies, but not siRNA delivered by Lipofectamine transfection, suggesting a role for RAB18 in siRNA delivery and intracellular trafficking. American Society of Gene & Cell Therapy 2022-04-02 /pmc/articles/PMC9035644/ /pubmed/35505960 http://dx.doi.org/10.1016/j.omtn.2022.04.003 Text en © 2022 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Original Article
Lu, Jiamiao
Swearingen, Elissa
Hardy, Miki
Collins, Patrick
Wu, Bin
Yuan, Eric
Lu, Daniel
Li, Chi-Ming
Wang, Songli
Ollmann, Michael
RAB18 is a key regulator of GalNAc-conjugated siRNA-induced silencing in Hep3B cells
title RAB18 is a key regulator of GalNAc-conjugated siRNA-induced silencing in Hep3B cells
title_full RAB18 is a key regulator of GalNAc-conjugated siRNA-induced silencing in Hep3B cells
title_fullStr RAB18 is a key regulator of GalNAc-conjugated siRNA-induced silencing in Hep3B cells
title_full_unstemmed RAB18 is a key regulator of GalNAc-conjugated siRNA-induced silencing in Hep3B cells
title_short RAB18 is a key regulator of GalNAc-conjugated siRNA-induced silencing in Hep3B cells
title_sort rab18 is a key regulator of galnac-conjugated sirna-induced silencing in hep3b cells
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9035644/
https://www.ncbi.nlm.nih.gov/pubmed/35505960
http://dx.doi.org/10.1016/j.omtn.2022.04.003
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