Multivalent N-Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing

Abstract

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.

The Role of micro RNAs in Breast Cancer Metastasis: Preclinical Validation and Potential Therapeutic Targets

“…Various chemically stabilized siRNAs that are conjugated on their 3' end with a trivalent GalNAc entity mediate great knockdown efficiencies of respective genes in hepatocytes. Thus, these conjugates are subject to several clinical trials against different indications such as amyloidosis, haemophilia and hypercholesterolemia (197)(198)(199).…”

RNA Therapeutics in Oncology: Advances, Challenges, and Future Directions

“…The discovery and development of N-acetyl galactosamine (GalNAc)-conjugated ASOs and RNA, siRNA, have enabled the preferential delivery of these agents to hepatocytes by virtue of the affinity of GalNAc for the hepatocyte-specific asialoglycoprotein receptors. [40][41][42][43] GalNAc conjugation of siRNAs has enabled moving away from complex lipid-nanoparticle delivery modalities, and GalNAcconjugated ASOs have increased in vivo potency of approximately 32-fold in the clinic compared to unconjugated parent ASOs. 44 The second major recent advance is the discovery of a next-generation class of RNAse H1 active ASOs that employ 2ʹ-4ʹ constrained ethyl (cEt).…”

“…[48][49][50][51][52][53][54] However, as mentioned briefly above, a more effective solution to the in vivo delivery of siRNA molecules (at least for the delivery of siRNA to hepatocytes) has been has been the conjugation of double-stranded siRNA molecules (and ASOs) to GalNAc moieties resulting in the active uptake of these conjugates into hepatocytes via Asialoglycoprotein receptors. 43 However, to achieve effective stability and distribution of GalNAcconjugated siRNAs, substantial additional chemical modifications must be incorporated, and thus, most GalNAc-conjugated siRNAs in development contain 10 to 14 PS moieties, 11 to 14 2ʹ-methoxy and 10 to 14 2ʹ-fluoro (2ʹ-F)-modified nucleosides. 55 The modifications employed in RNA therapeutic molecules can have important consequences both in the intact siRNA molecule and on metabolism.…”

A Highly Durable RNAi Therapeutic Inhibitor of PCSK9

“…These carbohydrates bind to abundant liver-expressed asialo-glycoprotein receptors, leading to inclisiran uptake specifically into hepatocytes. 16 The siRNA was modified with a combination of phosphorothioate, 2′-O-methyl nucleotide, and 2′-fluoro nucleotide modifications to improve molecular stability. 16 In preclinical studies involving non-human primates, doses of more than 3 mg per kilogram of body weight resulted in reductions of more than 80% in plasma PCSK9 levels and approximately 60% lowering of the serum LDL cholesterol level, with peak effects lasting more than 30 days, with a very slow return to baseline levels over a period of 90 to 120 days after administration (unpublished data).…”

“…16 The siRNA was modified with a combination of phosphorothioate, 2′-O-methyl nucleotide, and 2′-fluoro nucleotide modifications to improve molecular stability. 16 In preclinical studies involving non-human primates, doses of more than 3 mg per kilogram of body weight resulted in reductions of more than 80% in plasma PCSK9 levels and approximately 60% lowering of the serum LDL cholesterol level, with peak effects lasting more than 30 days, with a very slow return to baseline levels over a period of 90 to 120 days after administration (unpublished data). This phase 1 study assessed the safety, side-effect profile, and pharmacodynamic effects of inclisiran when it was administered subcutaneously in single or multiple doses in healthy volunteers who had an LDL cholesterol level of at least 100 mg per deciliter (2.60 mmol per liter) and in a small number of participants taking a stable dose of statin co-therapy.…”