Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates

Abstract

Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.

Figure 1.

Comparison siTTR-1 and siTTR-2 conjugates: (A) dose response in C57BL/6 female mice 4 days post a single SC administration; (B) metabolic stability in cynomolgus monkey plasma, rat liver tritosomes and rat liver cytosol; compounds were incubated with different matrices for 24 h and remaining full-length sense and antisense strands were quantified by HPLC.

Figure 2.

Concentration-time profiles of siTTR-1 and siTTR-2 in plasma (A), liver (B) and kidney (C) after a single SC or IV administration of 10 mg/kg in C57BL/6 male mice; shown are the individual data points (from two animals) at each time point as well as their mean (dotted lines). The insert in (B) shows the first 24 h post dose.

Figure 3.

Ttr mRNA knockdown as a function of time in mice treated with 10 mg/kg siTTR-1 or siTTR-2 SC relative to PBS-treated animals at 24 h and normalized to mouse Gapdh mRNA; shown are the individual data points (from two animals) at each time point as well as their mean (dotted lines).

Figure 4.

Time-concentration profiles of siAT-2 after a single SC dose of 2.5 mg/kg in mice overlaid with the gene silencing profile as measured by reduction of AT mRNA. siRNA concentration in (A) total liver and (B) RISC after Ago2 immunoprecipitation, measured by stem-loop RT-qPCR; shown are the individual data points (from two animals) at each time point as well as their mean (dotted lines).

Figure 5.

Silencing activity (as measured by reduction of AT mRNA in liver tissue) as a function of (A) siRNA concentration in total liver and (B) concentration of RISC-loaded siRNA.

Figure 6.

AT protein levels were measured following single SC injection of siAT-1 (10 mg/kg) or siAT-2 (1 and 10 mg/kg) in cynomolgus monkeys (n = 3 males per group). AT protein was measured in serum for ∼60 days and protein levels from individual animals were normalized to their respective individual pre-dose serum protein level at each time point. Each data point represents the remaining AT for the group average of three animal samples assayed in technical triplicates ± the standard deviation of the group.