Fig 1: Delivery of GalNAc-Conjugated ASOs to Tumor Xenografts(A) Schematic of the tumor xenograft model and ASO dosing schedule of tumor-bearing NSG mice. A total of 5 × 106 U87-H2b cells (control) and U87-H1a cells (active receptor) were subcutaneously implanted into the left and right flank of adult NSG mice, respectively. Three animals per group were treated with saline, control ASO, SMN-ASO, and GN3-SMN-ASO at 200 mg/kg/week, with two s.c. injections per week. Three days after the last injection, tumor, liver, and kidney samples were collected and processed for RNA, protein, and histological analysis. (B) Representative pictures of IHC analysis of ASGP-R1/2 expression and ASO localization in U87-H2b, U87-H1a, and liver sections after 2 weeks of ASO treatment. The rabbit anti-ASO antibody (Ionis Pharmaceuticals) recognizes the ASO’s phosphorothioate backbone. Scale bar, 100 µm. (C) Western blot analysis of U87-H1a and U87-H2b tumors confirms ASGP-R1 and ASGP-R2 expression. Cultured U87 cells expressing ASGP-R1 and ASGP-R2 express comparable receptor levels. (D) SMN2 exon 7 inclusion ratio in saline- and ASO-treated U87-H1 and U87-H2 control tumors was quantified by radioactive RT-PCR. (E) Quantification of results in (C). n = 3 animals/treatment; bar graphs represent mean ± SE. *p < 0.05; **p < 0.01 (Student’s t test). n.s., not significant.
Fig 2: Ectopic Expression of ASGP-R1 in U87 Cells Increases Efficacy of GN3-SMN-ASO In Vitro(A) Major and minor transmembrane domain (TM)-containing ASGP-R isoform cDNAs were cloned into retroviral and lentiviral expression vectors as indicated. The lentiviral vector carries the selection marker puromycin. ASGP-R H2c has a short 18-amino acid deletion in the intracellular domain of the receptor. (B) Western blot confirms expression of major and minor ASGP-R isoforms in U87 glioblastoma cells. The expression level of H1a is approximately 6-fold higher than the endogenous expression level in HepG2 cells, normalized to tubulin. H2a and H2b isoforms are detectable when expressed alone, but are stabilized in the presence of H1a. (C) Micrographs of U87 cells expressing ASGPR-H1a and H2b alone, and in combination. Cells were stained for ASGP-R1 (red), ASGP-R2 (green), and DAPI (blue). Arrowheads indicate non-uniform distribution of ASGP-R H2b, consistent with ER localization. Scale bar, 50 µm. (D) The SMN ASOs used in this study bind to intron 7 of SMN2 and promote exon 7 inclusion. Full-length SMN2 mRNA was quantified by radioactive RT-PCR; the product was digested with DdeI to separate SMN1 from SMN2 products. (E) U87 cells expressing major and minor ASGP-R isoforms alone or in combination were incubated with 300 nM unconjugated (SMN-MOE) or GalNAc-conjugated SMN-MOE ASOs (GN3-SMN-MOE) for 5 days by free uptake. Representative radiograph shows full-length SMN2 (top band) and SMN2 ? exon 7 (bottom band). (F) Quantification of full-length SMN2 in ASO-treated U87 cells. The differences among the means in the SMN group (p = 0.0055) and the GN3-SMN group (p < 0.0001) are statistically significant (one-way ANOVA). However, co-expression of H1a with H2b or H2c does not improve GN3-SMN-MOE uptake when compared with H1a alone (Student’s t test). n = 3 independent retroviral transductions; bar graphs represent mean ± SE. **p < 0.01. (G) U87 and U87-H1a cells exposed to unconjugated and GN3-conjugated SMN-ASOs for 24 h. Cells were stained for ASGP-R1 (red), ASO (green), and DAPI (blue). Arrows indicate ASGP-R1-expressing U87 cells, and arrowheads indicate ASGP-R1-negative cells. Scale bar, 50 µm. n.s., not significant; NTC, no-treatment control.
Fig 3: Ectopic Expression of ASGP-R in HepG2 Cells Increases GN3-ASO Activity in 3D Culture(A) Quantification of full-length SMN2 mRNA in HepG2 cells treated with 0–30 µM SMN and GN3-SMN-ASO for 5 days. (B) Western blots of stable 2D HepG2 cells expressing ASGP-R H1a and H2b alone or in combination. Tubulin served as a loading control. (C) Quantification of full-length SMN2 mRNA in 2D HepG2 cells treated with 300 nM SMN and GN3-SMN-ASOs for 5 days. Ectopic expression of ASGP-R1 and/or ASGP-R2 had no effect on ASO efficacy in 2D HepG2 cultures. p values for each group (one-way ANOVA) are indicated below the graph. (D) HepG2-WT (arrows) and HepG2-H1a cells (arrowheads) were cultured together and exposed to 300 nM SMN and GN3-SMN ASO for 24 h. Immunofluorescence staining shows ASGP-R1 (red), ASO (green), and nuclei (blue). HepG2-WT cells are ASGP-R1-positive, but the staining is very weak. Scale bar, 100 µm. (E) Phase-contrast micrographs of HepG2 cells grown attached on standard tissue culture plates (top) or in 3D cultures on ultra-low-attachment plates (bottom). Scale bars, 500 µM. (F) Western blots of stable 3D HepG2 cells expressing ASGP-R H1a and H2b alone or in combination. Tubulin served as a loading control. (G) Quantification of full-length SMN2 mRNA in 3D HepG2 cells treated with 300 nM SMN and GN3-SMN-ASOs for 5 days. Exon 7 inclusion upon GN3-SMN-ASO treatment is significantly greater in 3D HepG2 cells overexpressing ASP-R. p values for each group (one-way ANOVA) are indicated below the graph. n = 3 independent experiments; bar graphs represent mean ± SE. *p < 0.05; **p < 0.01 (Student’s t test). n.s., not significant; NTC, no-treatment control.
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