Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo

Abstract

Small interfering RNA (siRNA)-based drugs require chemical modifications or formulation to promote stability, minimize innate immunity, and enable delivery to target tissues. Partially modified siRNAs (up to 70% of the nucleotides) provide significant stabilization in vitro and are commercially available; thus are commonly used to evaluate efficacy of bio-conjugates for in vivo delivery. In contrast, most clinically-advanced non-formulated compounds, using conjugation as a delivery strategy, are fully chemically modified (100% of nucleotides). Here, we compare partially and fully chemically modified siRNAs in conjugate mediated delivery. We show that fully modified siRNAs are retained at 100x greater levels in various tissues, independently of the nature of the conjugate or siRNA sequence, and support productive mRNA silencing. Thus, fully chemically stabilized siRNAs may provide a better platform to identify novel moieties (peptides, aptamers, small molecules) for targeted RNAi delivery.

Figure 1.

Chemical composition and cellular efficacy of fully modified hsiRNAs. (A) Chemical structure, modification pattern, and molecular model of partially (hsiRNA) and fully modified (FM-hsiRNA) hydrophobic siRNA. (B and C) Comparison of hsiRNA and FM-hsiRNA activity in vitro. HeLa (B) or primary cytotrophoblasts (C) were incubated with hsiRNA or FM-hsiRNA at concentrations shown for 72h. mRNA levels were measured using QuantiGene (Affymetrix) normalized to housekeeping gene (human PPIB for (B) and human YWHAZ for (C)), and presented as percent of untreated control (n = 3, mean ± SD). UNT—untreated cells. (D, E) RISC loading and cleavage comparison of unmodified, naked-siRNA^LET7, or FM-hsiRNA^LET7 (D) FM-hsiRNA ^LET7 shows an increase in loaded RISC as evident by single turnover target cleavage. (E) Multiple turnover cleavage rate (normalized to loaded RISC) is similar between naked-siRNA^{, or FM-hsiRNA.}

Figure 2.

Systemically administered fully modified hsiRNA shows enhanced tissue distribution. Tissue distribution of Cy3-Chol-hsiRNA ^sFLT1 and Cy3-Chol-FM-hsiRNA ^sFLT after 10 mg/kg intravenous (IV) tail vein injection (A) or 10 mg/kg subcutaneous (SC) injection (B). Tissue distribution of Cy3-DHA-hsiRNA ^sFLT1 and Cy3-DHA-FM-hsiRNA ^sFLT after 10 mg/kg intravenous (IV) tail vein injection (C) or 10 mg/kg subcutaneous (SC) injection (D). Tissue distribution of Cy3-GalNAc-hsiRNA ^sFLT1 and Cy3-GalNAc-FM-hsiRNA ^sFLT after 10 mg/kg intravenous (IV) tail vein injection (E) or 10 mg/kg subcutaneous (SC) injection (F). Cy3-hsiRNA (red), nuclei stained with DAPI (blue). For every conjugate the left panels are tiled tissue images (scale bar = 1 mm) and the right panels are higher magnification tissue images (scale bar = 25 μm). Image is representative.

Figure 3.

Systemic administration of fully modified hsiRNAs shows enhanced tissue accumulation. Guide strand tissue quantification by PNA hybridization-based assay in tissues from Figure 2. Guide strand quantification of Cy3-Chol-hsiRNA ^sFLT1 and Cy3-Chol-FM-hsiRNA^sFLT after 10 mg/kg intravenous (IV) tail vein injection (A) or 10 mg/kg subcutaneous (SC) injection (B). Guide strand quantification of Cy3-DHA-hsiRNA ^sFLT1 and Cy3-DHA-FM-hsiRNA ^sFLT after 10 mg/kg intravenous (IV) tail vein injection (C) or 10 mg/kg subcutaneous (SC) injection (D). Guide strand quantification of Cy3-GalNAc-hsiRNA ^sFLT1 and Cy3-GalNAc-FM-hsiRNA ^sFLT after 10 mg/kg intravenous (IV) tail vein injection (E) or 10 mg/kg subcutaneous (SC) injection (F). Data presented as mean ± SD (n = 3 mice).

Figure 4.

Fully modified hsiRNAs are more efficacious than partially modified hsiRNAs following systemic administration. (A–C) Quantification of target (Ppib, Htt, sFlt1, respectively) mRNA silencing in liver 7 days after SC administration of cholesterol-conjugated ^vhsiRNA^NTC, ^vFM-hsiRNA^NTC, ^vhsiRNA^Target, ^vFM-hsiRNA^Target at 20 mg/kg. (D) Quantification of Ppib mRNA silencing in kidney 7 days after SC administration of cholesterol-conjugated ^vhsiRNA^NTC, ^vFM-hsiRNA^NTC, *hsiRNA, *FM-hsiRNA^sFlt1 at 20 mg/kg. FVBNj mice (n = 6 per group). mRNA levels were measured with QuantiGene 2.0 (Affymetrix) assay. Htt, Ppib, and sFlt1 mRNA levels normalized to housekeeping gene, Hprt. All data presented as percent of PBS treated control. All error bars represent mean ± SD. *P< 0.05, ***P< 0.001; ****P< 0.0001 as calculated by one-way ANOVA with Tukey's test for multiple comparisons. ^v = 5′-vinyl phosphonate, NTC = non-targeting control.