Effect of Free GalNAc Moiety and Linker Stability on AON-Mediated Cytotoxicity
(A and B) Exposing PTECs to free molecules of triGalNAc at the indicated concentrations did not lead to an apparent effect on EGF uptake after 6 days (A) and cell viability after 9 days (B). AON-C at 100 μM was used as toxic control. (C and D) Concentration-dependent effects of AON-A, AON-B, and AON-C on EGF uptake (C) and intracellular ATP (D) in PTECs in the absence or presence of equimolar concentrations of free GaNAc (triGalNAc), showing that the AON-induced cytotoxicity is not mitigated by exposure to free GalNAc molecules. Data represent means and SD (n = 3). An ANOVA with Sidak’s multiple comparisons test comparing GalNAc to Naked of the same AON and concentration indicates no significance for all conditions. (E) Gene expression analysis showing PCSK9 target gene knockdown in PTECs after 2 days of exposure to AON-B in its naked form or after conjugation to GalNAc via a cleavable versus an uncleavable linker. Data points represent means and SD (n = 3). ECs 50 are shown and indicate a further reduction of GalNAc-AON-B potency when coupled to GalNAc via an uncleavable linker compared to a cleavable linker. (F) Significant EGF accumulation in PTECs is observed after treatment with naked AON-B (p < 0.0001 at 10, 30, and 100 μM) but not with cleavable and uncleavable GalNAc AON-B. Data represent means and SD (n = 3); ANOVA with Dunnett’s multiple comparisons test.