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.

Diverse lipid conjugates for functional extra-hepatic siRNA deliveryin vivo

“…The siRNAs used in these studies were fully chemically modified for maximal stability and minimal innate immune activation as described (see Figure 1a and Methods). We and others have recently shown that full chemical stabilization is essential for conjugated-mediated delivery [19][20][21] . The chemical compositions of lipids had a profound effect on the hydrophobicity of lipid-siRNA, based on retention time in reversed-phase HPLC 36 , which ranged from 1 to 12.2 minutes (Figure 1b).…”

“…Thus, full chemical stabilization of siRNAs is essential for conjugate-mediated delivery 19 . The clinical success of GalNAc conjugated siRNAs was only achieved when the siRNAs are extensively modified 20,21 .…”

Pharmacodynamic and Pharmacokinetic Properties of Full Phosphorothioate Small Interfering RNAs for Gene Silencing In Vivo

“…Although our understanding of the cellular uptake of therapeutic oligonucleotides is limited, siRNAs are thought to be transported in endosomal vesicles that are subsequently fused with lysosomes [46][47][48]. While several nanoparticle or polymer based formulations have been developed to facilitate endosomal escape [49], nuclease stability during endosomal trafficking is required for the administration of oligonucleotides formulated in PBS and has been identified as a key determinant of the in vivo efficacy of GalNAc-conjugated siRNAs [15]. Rat liver tritosomes are an in vitro surrogate for cellular lysosomal compartments [50].…”

“…However, givosiran is administered as a nanoparticle-free solution. For this purpose, this siRNA relies on complex chemical modification of the natural oligoribonucleotide in three main structural ways: (1) modification of every nucleoside with 2¢-OMe or 2¢-F moieties and terminal phosphorothioate (PS) linkages, (2) conjugation through a binary linker to (3), a trivalent Nacetylgalactosamine ligand for targeted delivery to hepatocytes [15]. The structural complexity of such molecules poses an additional challenge in terms of metabolite characterization and risk assessment with respect to long-term exposure.…”

Selective tissue targeting of synthetic nucleic acid drugs

“…The GalNAc-siRNA conjugates were rapidly cleared from plasma into hepatocytes, and and excess GalNAc ligand could compete with this uptake, showing specificity of ASGR-mediated uptake. Recent studies highlighted the importance of chemical modification to the GalNAc-siRNA conjugates to enhance potency and duration of effect (90). Chemical modifications help siRNA survive nucleolytic degradation as it transits through endo-lysosomal compartments before release to the cytosol.…”