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. 2022 Apr 2:28:423-434.
doi: 10.1016/j.omtn.2022.04.003. eCollection 2022 Jun 14.

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

Jiamiao Lu  1 , Elissa Swearingen  1 , Miki Hardy  1 , Patrick Collins  1 , Bin Wu  1 , Eric Yuan  1 , Daniel Lu  1 , Chi-Ming Li  1 , Songli Wang  1 , Michael Ollmann  1
Affiliations

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

Jiamiao Lu et al. Mol Ther Nucleic Acids. .

Abstract

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.

Keywords: GalNAc conjugates; MT: RNA/DNA editing; RAB18; genome-wide; knockout screen; siRNA efficacy; siRNA trafficking.

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Conflict of interest statement

All authors have the following conflicts of interest to declare: J.L., E.S., B.W., E.Y., D.L., C.-M.L., and S.W. are employees of Amgen Inc. M.O., P.C., and M.H. 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.

Figures

None
Graphical abstract
Figure 1
Figure 1
Validation of screen conditions for genome-wide pooled CRISPR-Cas9 screen (A) Comparison of target gene (ASGR1) silencing potency in human primary hepatocytes, Hep3B and HepG2 cells by two GalNAc-conjugated ASGR1 siRNAs. The features of these two siRNA conjugates are described in Table S2. (B) Treatment with an in-house-made anti-ASGR1 antibody, 7E11, mitigated the HPRT1 gene silencing induced by GalNAc-HPRT1 siRNA (8172) in Hep3B cells. The left panel outlines the experiment scheme, and the right panel shows the ddPCR measurement of HPRT1 mRNA levels in percentage normalized by housekeeping gene TBP (TATA box binding protein) readings and no siRNA (PBS only) treated control group. The feature and sequence of siRNA 8172 is described in Table S2. (C) Dose-dependent kill curve of 6TG treatment in Hep3BCas9 cells. (D) A small-scale pilot experiment to validate the feasibility of using HPRT1-6TG live/dead selection for CRISPR screen. The gRNA lentivirus library transduced Hep3BCas9 cells were treated with GalNAc-HPRT1 siRNA and/or 6TG (100 μL) in different groups. The viable cell count measured by ViCell on day 3 and day 6 after 6TG treatment for each treatment group was normalized by negative control group readings. The resulting normalized viability percentage of each group at both time points was plotted into bar graph. Left panel: day 3 post-6TG treatment data. Right panel: day 6 post-6TG treatment data. Error bars indicate SD (standard deviation) of three replicates.
Figure 2
Figure 2
Large-scale genome-wide pooled CRISPR-knockout screen experiment and candidate gene validation (A) Experiment scheme of large-scale genome-wide pooled CRISPR-knockout screen. (B) Analysis of the CRISPR screen results by overlapping enriched genes in both 150 nM siRNA + 6TG treated samples (150si6TGd9) versus no siRNA but 6TG treated samples (nosi6TGd9) and 750 nM siRNA + 6TG treated samples (750si6TGd9) versus no siRNA but 6TG treated samples (nosi6TGd9). A total of 17 genes were identified with FDR < 0.2 (outlined by dashed line). (C) Analysis of the CRISPR screen results by overlapping enriched genes from 750 nM siRNA + 6TG treated samples (750si6TGd9) versus no siRNA but 6TG treated samples (nosi6TGd9) with depleted genes in 6TG only vs no siRNA no 6TG samples. The horizontal axis indicates the sensitivity to 6TG. The dashed line outlines 8 genes with FDR < 0.2 that were heavily depleted upon 6TG treatment. (D) Experiment scheme for testing of regulators of HPRT1 siRNA activity using secondary arrayed multiplexed synthetic gRNA screening in 96-well format. (E) Heatmap results of secondary arrayed multiplexed synthetic gRNA shown in (C). In the heatmap, red indicates reduced HPRT1 siRNA silencing activity, and blue indicates enhanced HPRT1 siRNA silencing activity. Colors indicate the percentage of HPRT1/TBP mRNA signals detected through ddPCR and normalized to no siRNA control.
Figure 3
Figure 3
Validation of the effect of RAB18 knockdown and knockout on siRNA silencing potency (A) Twenty-four hour knockdown efficacy of three siRNA molecules targeting RAB18 in Hep3B cells. Plotted are the ddPCR measured RAB18 mRNA levels. (B) The ddPCR measurement of RAB18 mRNA levels in the cells used for analysis described in (C) on day 5 after siRAB18 and siNTC treatment, or day 4 after GalNAc-HPRT1 siRNA treatment. Error bars indicate SD of three replicates. (C) Measurement of GalNAc-HPRT1 siRNA silencing potency in both siRAB18_3 and siNTC-treated Hep3B cells by ddPCR on day 4 after GalNAc-HPRT1 siRNA treatment. Plotted are the ddPCR-measured HPRT1 mRNA levels. (D) HPRT1-6TG live/dead selection performed in both Hep3BCas9 and RAB18-knockout cells. The left panel outlines the experiment scheme, and the right panel shows the cell death rate measured with CellTiter-Glo reagents (Promega, Madison, WI). (E) Measurement of GalNAc-HPRT1 siRNA silencing potency in both Hep3BCas9 and RAB18-knockout cells by ddPCR on day 4 after siRNA treatment. The left panel summarizes the experiment scheme, and the right panel shows the ddPCR-measured HPRT1 mRNA levels. PPIB siRNA was used as control siRNA. (F) Measurement of GalNAc-ASGR1 siRNA silencing potency in both Hep3BCas9 and RAB18-knockout cells by ddPCR on day 4 post siRNA treatment. The experiment scheme is the same as shown in (E). Plotted here are ASGR1 mRNA levels measured by ddPCR. PPIB siRNA was used as control siRNA. (G) The same experiment shown in (E) was performed using GalNAc-PPIB siRNA. The left panel lists the expression profiles of target genes in FPKM (fragments per kilobase of transcript per million mapped reads) (obtained from Broad Institute Cancer Cell Line Encyclopedia [CCLE]). The right panel plots the ddPCR measurement of PPIB mRNA levels. HPRT1 siRNA was used as control siRNA. (H) Antibody-blocking test in Hep3BCas9 and RAB18-knockout cells by using anti-ASGR1 antibody 7E11. The left panel summarizes the experiment scheme, and the right panel shows the ddPCR measurement of HPRT1 mRNA levels. (I) Unconjugated HPRT1 siRNA transfection assay in Hep3BCas9 and RAB18-knockout cells. Plotted are the ddPCR-measured HPRT1 mRNA levels on day 4 after siRNA treatment. All ddPCR results shown were normalized by TBP and no siRNA (PBS only) treated control group.
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