Figure 1.

GalNAc AONs Are Less Toxic than Naked AONs in Renal Tubular Cells In Vitro

(A) Chemical structure of a GalNAc-AON: 5′-phosphodiester bound (red) of an AON (blue) is coupled to three GalNAc groups (green) via a triantennary linker (black). The 5′-ca (blue) is cleavable. The underlined AON sequence corresponds to the naked AON. (B) Schematic representation of in vitro assay design for AON nephrotoxicity profiling using human immortalized PTECs (PTEC-TERT). Accumulation of EGF in supernatant is detected by ELISA and indicates decreased cell functionality. Reduction of intracellular ATP indicates reduced cell viability. (C and D) Measurement of EGF in the supernatant of PTECs treated for 6 days (C) and measurement of intracellular ATP of PTEC treated for 9 days (D) with a panel of AONs in their Naked or GalNAc form at 100 μM, revealing a generally reduced effect of GalNAc AONs. Data represent means and SD (n = 3). In both assays, statistical significance over control was observed for Naked AONs 1, 2, 5, 7, 9, 10, 16, and 18 (p < 0.005) and GalNAc-AON 7 (p < 0.02); ANOVA with Dunnett’s multiple comparisons test. (E) Concentration-dependent EGF profile of the known nephrotoxic AON-B showing abolition of cytotoxic effect by GalNAc conjugation of AON-B. Data represent means and SD (n = 3).