Ligand conjugate SAR and enhanced delivery in NHP

Abstract

N-Acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) are a leading RNA interference (RNAi) platform allowing targeted inhibition of disease-causing genes in hepatocytes. More than a decade of development has recently resulted in the first approvals for this class of drugs. While substantial effort has been made to improve nucleic acid modification patterns for better payload stability and efficacy, relatively little attention has been given to the GalNAc targeting ligand. In addition, the lack of an intrinsic endosomal release mechanism has limited potency. Here, we report a stepwise analysis of the structure activity relationships (SAR) of the components comprising these targeting ligands. We show that there is relatively little difference in biological performance between bi-, tri-, and tetravalent ligand structures while identifying other features that affect their biological activity more significantly. Further, we demonstrate that subcutaneous co-administration of a GalNAc-functionalized, pH responsive endosomal release agent markedly improved the activity and duration of effect for siRNA conjugates, without compromising tolerability, in non-human primates. These findings could address a significant bottleneck for future siRNA ligand conjugate development.

Keywords: ASGPr; GalNAc; endosomal release; siRNA.

Graphical abstract

Figure 1

Schematic of GalNAc ligand, geometry and interaction with ASGPr (A) GalNAc conjugate structural nomenclature. (B) Schematic representation of the individual ligand elements aligning with trimeric carbohydrate recognition domains of ASGPR.

Figure 2

Activity and tolerability of GalNAc conjugates ± polymer micelle in mice (A) Dose response study: C57BL/6 mice (n = 4) were treated with a single subcutaneous dose of either 1 or 3 mg/kg GalNAc-siRNA alone, or in combination with 10 mg/kg polymer (n.s. = p > 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two-way ANOVA analysis). (B) Repeat dose study: C57BL/6 mice (n = 4) were treated with subcutaneous administrations of 2 mg/kg conjugate alone or in combination with 10 mg/kg polymer, every 3 weeks (as indicated by arrows), for a period of 18 weeks. TTR protein concentration in plasma was determined using ELISA and reported as a percentage of pre-dose saline control group mean (n.s. = p > 0.05, ∗∗p < 0.01, two-way ANOVA analysis). (C) Repeat dose study: C57BL/6 mice (n = 4) were monitored for body weight changes after repeated subcutaneous administrations of 2 mg/kg conjugate alone or in combination with 10 mg/kg polymer (n.s. = p > 0.05, two-way ANOVA analysis). (D) Polymer titration study: C57BL/6 mice (n = 5) were treated with a single subcutaneous dose of polymer micelle at the specified dose levels. At 6 h post-dose, mouse plasma samples were collected for determination of cytokine levels by ELISA (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; one-way ANOVA analysis with Dunnett’s multiple comparison test when comparing with PBS control group). Data are presented as mean ± SEM.

Figure 3

Activity and tolerability of GalNAc conjugates ± polymer micelle in NHPs (A) A single subcutaneous injection of bivalent or tetravalent GalNAc-siRNA was administered to male cynomolgus monkeys (Macaca fascicularis) at 3 mg/kg. In one treatment group, 0.6 mg/kg tetravalent GalNAc-siRNA was co-administered with 8.8 mg/kg polymer micelle. Blood samples were processed to serum and analyzed with a quantitative biomarker immunoassay. Serum TTR levels of each animal were normalized to its respective baseline level at pre-dose. Group average at each time point is represented as a percentage relative to its pre-dose group average (n.s = p > 0.05, ∗∗∗p < 0.001, two-way ANOVA analysis). (B) A single subcutaneous injection of 3 mg/kg tetravalent GalNAc-siRNA, 0.6 mg/kg tetravalent GalNAc-siRNA + 8.8 mg/kg polymer, or 8.8 mg/kg polymer alone was administered to male cynomolgus monkeys (Macaca fascicularis). Blood samples were collected at 6 h post-dose, processed to plasma, and analyzed with a quantitative, multiplexed inflammatory biomarker assay (n.s = p > 0.05, one-way ANOVA analysis). Data are presented as mean ± SEM (n = 3 per group).