RAB18 regulates extrahepatic siRNA-mediated gene silencing efficacy

Abstract

Small interfering RNAs (siRNAs) hold considerable therapeutic potential to selectively silence previously "undruggable" disease-associated targets, offering new opportunities to fight human diseases. This therapeutic strategy, however, is limited by the inability of naked siRNAs to passively diffuse across cellular membranes due to their large molecular size and negative charge. Delivery of siRNAs to liver through conjugation of siRNA to N-acetylgalactosamine (GalNAc) has been a success, providing robust and durable gene knockdown, specifically in hepatocytes. However, the poor delivery and silencing efficacy of siRNAs in other cell types has hindered their applications outside the liver. We previously reported that a genome-wide pooled knockout screen identified RAB18 as a major modulator of GalNAc-siRNA conjugates. Herein, we demonstrate RAB18 knockout/knockdown efficaciously enhances siRNA-mediated gene silencing in hepatic and extrahepatic cell lines and in vivo. Our results reveal a mechanism by which retrograde Golgi-endoplasmic reticulum (ER) transport and the intracellular lipid droplets (LDs) positively regulate siRNA-mediated gene silencing.

Keywords: MT: Oligonucleotides: Therapies and Applications; RAB18; extrahepatic; lipid droplets; retrograde Golgi-ER transport; siRNA; siRNA trafficking.

Graphical abstract

Figure 1

Impact of RAB18 gene expression on the silencing efficacy of siRNA and ASO molecules (A) Effect of RAB18 overexpression on siRNA-mediated gene silencing in Hep3BCas9 cells. The left and middle figures show flow cytometry data demonstrating the successful overexpression of the RAB18 gene using a lentivirus vector (Figure S1A). Cells overexpressing RAB18 are mCherry positive. The figure on the right shows dPCR measurements of HPRT1 expression in GalNAc-HPRT1 siRNA-treated Hep3BCas9 cells and Hep3BCas9RAB18_OE cells (n = 3). (B) Effect of RAB18 overexpression on siRNA-mediated gene silencing in Hep3BRABKO cells. The left and middle figures show the flow cytometry data, which demonstrate the successful overexpression of the RAB18 gene in Hep3BRAB18KO cells. The right figure shows dPCR measurements of HPRT1 expression in GalNAc-HPRT1 siRNA-treated Hep3BRAB18KO cells and the RAB18-overexpression Hep3BRAB18KO_OE cells (n = 3). (C) dPCR measurements of HPRT1 expression in Hep3BCas9 and Hep3BRAB18KO cells treated with GalNAc-conjugated ASO molecules targeting the HPRT1 gene (15469, 15470) (n = 3). All samples were measured 4 days post siRNA treatments. All dPCR results were normalized by the control housekeeping gene, TBP, and no siRNA (PBS only)-treated control groups. All error bars represent standard deviation (SD).

Figure 2

Impact of RAB18 knockout on siRNA-mediated gene silencing in extrahepatic cells (A) dPCR measurements of HPRT1 expression in Hep3BCas9, Hep3BRAB18KO, T47DCas9, and T47DRAB18KO cells treated with unconjugated HPRT1 siRNA (39986) (n = 4). (B) Editing efficacy on the RAB18 gene assessed by ICE analysis. (C) Editing efficacy on the RAB18 gene analyzed by western blot of extrahepatic cell lines. (D–I) dPCR measurements of HPRT1 expression in RAB18-knockout extrahepatic cell lines and their control cell lines (Cas9 only) treated with unconjugated HPRT1 siRNA or unconjugated non-targeting (NT) siRNA. All samples were measured 4 days post siRNA treatments. All dPCR results shown were normalized by TBP and no-siRNA (PBS only)-treated control groups. (J) dPCR measurements of RAB18 gene expression 3 days post RAB18 siRNA and NT siRNA transfection in human iPSC-derived cardiomyocytes. (K) dPCR measurements of HPRT1 gene expression of human iPSC-derived cardiomyocytes transfected with RAB18 siRNA or NT siRNA followed by treatment of GalNAc-HPRT1 siRNA (7 days post RAB18 siRNA and NT siRNA transfection). Asterisk indicates significant difference (∗p < 0.05); ns, not significant based on one-way ANOVA test. All error bars represent SD.

Figure 3

Results from the in vivo study evaluating the silencing effect of GalNAc-Rab18 siRNA triggers and transcriptomic analysis of liver RNA samples harvested from siRNA-treated mice (A) Schematic diagram illustrating the experimental workflow for the in vivo study. (B) dPCR measurements of Hprt1 expression in liver RNA samples harvested 14 days post -s.c. injection. (C) dPCR measurements of Rab18 expression in liver RNA samples harvested 14 days post s.c. injection. All dPCR results were normalized by Tbp and the control vehicle (PBS)-treated control groups. (D) Number of significant differentially expressed genes across all experimental groups as compared to vehicle control. Genes with a p-adjusted <0.05 and a fold-change >1.5 (up) or < −1.5 (down) were considered significantly differentially expressed for all panels. (E) Volcano plot of differentially expressed genes in 64131-1-treated samples compared with vehicle-treated control group. The numbers of significantly downregulated (blue) or upregulated - (red) genes are indicated at the top. (F) Top enriched Gene Ontology (GO) biological processes gene sets among upregulated genes from (E). (G) Top enriched GO biological processes gene sets among downregulated genes from (E). (H) Venn diagram and list of shared upregulated genes identified in all Rab18 siRNA trigger-treated samples compared to vehicle control. (I) Venn diagram and list of shared downregulated genes identified in all Rab18 siRNA trigger-treated samples compared to vehicle control. The adjusted p-value is calculated using the Benjamini-Hochberg method.

Figure 4

Results from the second in vivo study evaluating the effect of Rab18 knockdown on siRNA-mediated gene silencing (A) Schematic diagram illustrating the experimental workflow and group design for the second in vivo study. (B) dPCR measurements of Rab18 expression in liver RNA samples harvested 28 days post s.c. injection. (C) dPCR measurements of Hprt1 expression in liver RNA samples harvested 28 days post s.c. injection. All dPCR results were normalized by Tbp and the control vehicle (PBS)-treated control groups. Asterisk indicates significant difference (∗p < 0.05) based on one-way ANOVA test. All error bars represent SD.

Figure 5

Impact of RAB18 knockout on intracellular siRNA accumulation and the impact of retrograde Golgi-ER transport on siRNA-mediated gene silencing (A) Quantification of intracellular siRNA spots measured from imaging at various time points post GalNAc-HPRT1 siRNA treatment. (B) Schematic diagram of experimental design to evaluate the impact of retrograde Golgi-ER transport on siRNA-mediated gene silencing. (C) dPCR measurements of HPRT1 expression in Hep3BCas9 or Hep3BRAB18KO cells treated with GalNAc-HPRT1 siRNA (8172) in the presence or absence of GCA (n = 3). (D) dPCR measurements of HPRT1 expression in Hep3BCas9 or Hep3BRAB18KO cells transfected (Lipofectamine) with unconjugated HPRT1 siRNA (39986) in the presence or absence of GCA (n = 3). The unconjugated HPRT1 siRNA was directly added to culture medium for groups without Lipofectamine labels. All dPCR samples were measured 4 days post siRNA treatments. All dPCR results shown were normalized by TBP and no siRNA (PBS only)-treated control groups. Asterisk indicates significant difference (∗∗ p < 0.01) based on one-way ANOVA test. All error bars represent SD.

Figure 6

Imaging intracellular siRNA (A) The table on the left lists the antibodies and corresponding cellular markers evaluated by imaging. On the right, confocal microscopy images show siRNA co-staining with LipidTox (images of the other cellular markers listed are shown in Figures S6 and S7). Scale bars represent 5 μm. (B) Manders coefficient analysis of the images taken from Hep3BCas9 and He3BRAB18KO cells treated with 1.5 μM GalNAc-HPRT1 siRNA 2 days post siRNA treatment. The Manders coefficient scores reflect the frequency of siRNA colocalization with the given cellular markers. Compared to all other cellular markers, notable siRNA colocalization difference between Hep3BCas9 and Hep3BRAB18KO cells was detected for LipidTox-stained LDs. Each imaging experiment was repeated five times. (C) Pearson coefficient analysis on the refractive and fluorescence images of live Hep3BCas9 and He3BRAB18KO cells treated with 1.5 μM AF647 conjugated GalNAc-HPRT1 siRNA 1 and 2 days post siRNA treatment. Each imaging experiment was repeated six times. (D) Quantification of LD count on day 1 and day 2 post 1.5 μM GalNAc-HPRT1 siRNA treatment comparing Hep3BCas9 and Hep3BRAB18KO cells. Each imaging experiment was repeated eight times. (E) Summary of LD size analysis on day 1 or day 2 post 1.5 μM GalNAc-HPRT1 siRNA treatment comparing Hep3BCas9 and Hep3BRAB18KO cells. Each imaging experiment was repeated eight times. All error bars represent SD. Asterisk indicates significant difference (∗ p < 0.05; ∗∗ p < 0.01) based on one-way ANOVA test.

Figure 7

The effects of oleic acid and triacsin C on regulating siRNA-mediated gene silencing and the prerequisite of RAB18 knockout/knockdown in enhancing siRNA-mediated gene silencing dPCR measurements of HPRT1 expression in (A) Hep3BCas9 cells or (B) Hep3BRAB18KO cells treated with GalNAc-HPRT1 siRNA and triacsin C. Cells were pre-treated with the indicated concentration of triacsin C for 24 h prior to siRNA treatment. dPCR measurements of HPRT1 expression in (C) Hep3BCas9 cells or (D) Hep3BRAB18KO cells treated with GalNAc-HPRT1 siRNA and oleic acid. Cells were treated with the indicated concentration of oleic acid for 24 h prior to siRNA treatment. (E) dPCR measurements of HPRT1 expression in GalNAc-HPRT1 siRNA-treated Hep3BCas9 and Hep3BRAB18KO cells with or without co-treatment of GalNAc-RAB18 siRNA or the negative control GalNAc-PPIB siRNA. All dPCR samples were measured 4 days post siRNA treatments. All dPCR results shown were normalized by TBP and no-siRNA (PBS only)-treated control groups with n = 3 for each experiment. All error bars represent SD.