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

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.

Southern pine beetle-specific RNA interference exhibits no effect on model nontarget insects

The efficacy and high specificity of the RNA interference pathway has prompted its exploration as a potential molecular management tool for many insect pests, including the destructive southern pine beetle, Dendroctonus frontalis Zimmermann, in which gene knockdown and mortality via double-stranded RNAs (dsRNAs) have already been demonstrated in the laboratory. The nucleotide sequence of dsRNAs requires an exact match of at least 16 nucleotides with the targeted messenger RNA to trigger knockdown of that gene. This allows vital genes in a target pest to be silenced and mortality induced while reducing the probability of adverse effects in nontarget organisms. However, prior to utilization in forest ecosystems, demonstration of the specificity of dsRNAs through laboratory bioassays evaluating potential nontarget effects on model insects is required for proper risk assessment analyses. Consequently, we evaluated three SPB-specific dsRNAs for lethal effects, sublethal effects (larval growth rate, adult emergence or adult fecundity), and relative gene expression in three model nontarget insects representing key functional guilds, including a predator, herbivore, and pollinator. The SPB-specific dsRNAs had no effect on survival of our nontarget insects. Additionally, no sublethal effects were found and the gene expression analyses corroborated bioinformatic analyses in finding no gene knockdown. Our findings support the high specificity of RNAi technology and provide support for its development and deployment for protection of conifer forests against SPB with minimal nontarget concerns.

Haemonchus contortus Transthyretin-Like Protein TTR-31 Plays Roles in Post-Embryonic Larval Development and Potentially Apoptosis of Germ Cells

Transthyretin (TTR)-like proteins play multi-function roles in nematode and are important component of excretory/secretory product in Haemonchus contortus. In this study, we functionally characterised a secretory transthyretin-like protein in the barber’s pole worm H. contortus. A full-length of transthyretin-like protein-coding gene (Hc-ttr-31) was identified in this parasitic nematode, representing a counterpart of Ce-ttr-31 in Caenorhabditis elegans. High transcriptional levels of Hc-ttr-31 were detected in the egg and early larval stages of H. contortus, with the lowest level measured in the adult stage, indicating a decreased transcriptional pattern of this gene during nematode development. Localisation analysis indicated a secretion of TTR-31 from the intestine to the gonad, suggesting additional roles of Hc-ttr-31 in nematode reproduction. Expression of Hc-ttr-31 and Ce-ttr-31 in C. elegans did not show marked influence on the nematode development and reproduction, whereas Hc-ttr-31 RNA interference-mediated gene knockdown of Ce-ttr-31 shortened the lifespan, decreased the brood size, slowed the pumping rate and inhibited the growth of treated worms. Particularly, gene knockdown of Hc-ttr-31 in C. elegans was linked to activated apoptosis signalling pathway, increased general reactive oxygen species (ROS) level, apoptotic germ cells and facultative vivipary phenotype, as well as suppressed germ cell removal signalling pathways. Taken together, Hc-ttr-31 appears to play roles in regulating post-embryonic larval development, and potentially in protecting gonad from oxidative stress and mediating engulfment of apoptotic germ cells. A better knowledge of these aspects should contribute to a better understanding of the developmental biology of H. contortus and a discovery of potential targets against this and related parasitic worms.