ABSTRACT
Small interfering RNAs (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 due to steps required for endosomal escape, intracellular trafficking, and stability of siRNA. To reveal the cellular factors limiting delivery of siRNA therapeutics, we performed a pooled, genome wide knockout screen based on delivery of GalNAc conjugated siRNA targeting the HPRT1 gene in the human hepatocellular carcinoma line Hep3B. Our primary pooled genome wide knockout screen identified candidate genes that when knocked out significantly enhanced siRNA efficacy in Hep3B cells. Follow-up studies indicate that knockout of one gene in particular, RAB18, improved siRNA efficacy.
Competing Interest Statement
All authors have the following conflicts of interest to declare. Jiamiao Lu, Elissa Swearingen, Bin Wu, Eric Yuan, Daniel Lu, Chi-Ming Li, and Songli Wang are employees at Amgen Inc. Michael Ollmann, Patrick Collins, and Miki Hardy were employed by Amgen Inc. while working on the study. All authors owned Amgen shares when the study was carried out. However, these do not alter the authors' adherence to all journal policies on sharing data and materials. None of the authors serves as a current Editorial Team member for this journal.
