Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA−GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED 50 ) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA−GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA−GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.

In recent years, RNA interference (RNAi) therapeutics, most notably with lipid nanoparticle-based delivery systems, have advanced into human clinical trials. The results from these early clinical trials suggest that lipid nanoparticles (LNPs), and the novel ionizable lipids that comprise them, will be important materials in this emerging field of medicine. A persistent theme in the use of materials for biomedical applications has been the incorporation of biodegradability as a means to improve biocompatibility and/or to facilitate elimination. Therefore, the aim of this work was to further advance the LNP platform through the development of novel, next-generation lipids that combine the excellent potency of the most advanced lipids currently available with biodegradable functionality. As a representative example of this novel class of biodegradable lipids, the lipid evaluated in this work displays rapid elimination from plasma and tissues, substantially improved tolerability in preclinical studies, while maintaining in vivo potency on par with that of the most advanced lipids currently available.

Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.

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Asialoglycoprotein receptor (ASGPR) mediated delivery of triantennary N-acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) to hepatocytes is a promising paradigm for RNAi therapeutics. Robust and durable gene silencing upon subcutaneous administration at therapeutically acceptable dose levels resulted in the advancement of GalNAc-conjugated oligonucleotide-based drugs into preclinical and clinical developments. To systematically evaluate the effect of display and positioning of the GalNAc moiety within the siRNA duplex on ASGPR binding and RNAi activity, nucleotides carrying monovalent GalNAc were designed. Evaluation of clustered and dispersed incorporation of GalNAc units to the sense (S) strand indicated that sugar proximity is critical for ASGPR recognition, and location of the clustered ligand impacts the intrinsic potency of the siRNA. An array of nucleosidic GalNAc monomers resembling a trivalent ligand at or near the 3' end of the S strand retained in vitro and in vivo siRNA activity, similar to the parent conjugate design. This work demonstrates the utility of simple, nucleotide-based, cost-effective siRNA-GalNAc conjugation strategies.