Expanding Conjugate Space of RNAi Therapeutics: Ligand at the 3' End of the Antisense Strand Achieves Uncompromised In Vivo Potency and Efficacy and Reveals Interactions with the Argonaute-2 PAZ Domain.

The conjugation of the sense strands of small interfering RNA (siRNA) to tri--acetylgalactosamine (GalNAc), the ligand for a hepatocyte-specific receptor, enables the delivery of multiple clinically approved therapeutic agents that act through the RNA interference pathway. Here, we report the systematic evaluation of siRNAs with the 3' termini of antisense strands conjugated to GalNAc for the first time. These designs retained the same receptor affinity, in vitro and in vivo activities, as well as the same level of loading into the RNA-induced silencing complex as siRNAs with a GalNAc-conjugated sense strand.

Improved Metabolic Stability and Silencing Efficacy of siRNAs with Phosphorothioate Linkage-Free, GalNAc-Conjugated Sense Strands Containing Morpholino-LNA Modifications.

To ensure specificity, loading of the sense strand of small interfering RNAs (siRNAs) into RISC must be inhibited. We show here that siRNAs with 5'- and 6'-morpholino LNA residues or 6'-OH-LNA at the 5' terminus of a fully phosphodiester sense strand resulted in metabolically stable siRNAs with a potency and a duration of action in mice that were greater than those of an siRNA in which the 5' terminus of the sense strand has two terminal phosphorothioate linkages and regular LNA.

siRNAs containing 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2'-F-NMC NTPs.

We recently reported the synthesis of 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold.

Chimeric siRNAs with chemically modified pentofuranose and hexopyranose nucleotides: altritol-nucleotide (ANA) containing GalNAc-siRNA conjugates: in vitro and in vivo RNAi activity and resistance to 5'-exonuclease.

In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with the 2'-deoxy-2'-fluoro and 2'-O-methyl pentofuranose chemical modifications. The siRNAs were designed to silence the transthyretin (Ttr) gene and were conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Sense and antisense strands of the parent duplex were synthesized with single ANA residues at each position on the strand, and the resulting siRNAs were evaluated for their ability to inhibit Ttr mRNA expression in vitro.

Reversal of siRNA-mediated gene silencing 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.