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.

One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc–siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc–siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.

We report rapid, potent reversal of GalNAc-siRNA-mediated RNA interference (RNAi) activity in vivo with short, synthetic, high-affinity oligonucleotides complementary to the siRNA guide strand. We found that 9-mers with five locked nucleic acids (LNAs) have the highest potency across several targets. Our modular, sequence-specific approach, named REVERSIR, may enhance the therapeutic profile of any long-acting GalNAc-siRNA (short interfering RNA) conjugate by enabling control of RNAi pharmacology.

Malaria parasite erythrocytic stages comprise of repeated bursts of parasites via cyclical invasion of host erythrocytes using dedicated receptor-ligand interactions. A family of erythrocyte-binding proteins from Plasmodium knowlesi (Pk) and Plasmodium vivax (Pv) attach to human Duffy antigen receptor for chemokines (DARC) via their Duffy binding-like domains (DBLs) for invasion. Here we provide a novel, testable and overarching interaction model that rationalizes even contradictory pieces of evidence that have so far existed in the literature on Pk/Pv-DBL/DARC binding determinants. We further address the conundrum of how parasite-encoded Pk/Pv-DBLs recognize human DARC and collate evidence for two distinct DARC integration sites on Pk/Pv-DBLs.

Matrix metalloproteinases (MMPs) are a large family of calcium-dependent zinc- containing endopeptidases, which are responsible for the tissue remodeling and degradation of the extracellular matrix (ECM), including collagens, elastins, gelatin, matrix glycoproteins, and proteoglycan. The inappropriate expression of these MMPs constitutes part of the pathogenic mechanism in several diseases, therefore they are subject to inhibition. They can be inhibited by endogenous proteinase inhibitors such as 2-macroglobulin or by the family of tissue inhibitors of metalloproteinases (TIMPs), which are glycoproteins of molecular weight 21-30 kDa, consisting of 184-194 amino acid residues. Recently, many different classes of synthetic inhibitors have been developed in which the hydroxamic acidbased class of compounds (hydroxamates) have been most widely studied, as their hydroxamic acid group (CONHOH) enables them to act as a bidentate ligand with the zinc ion present in MMPs, leading to much stronger interaction with the receptor as compared to any other class of inhibitors. The present review describes in detail the recent development on this class of MMP inihibitors. Compounds like 12,17e, f, g and h, 45j, 45k, 50f, 62a, 63a, and 63b have been reported to be highly promising for further development.