GalNAc-siRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics

Abstract

Short-interfering RNA (siRNA)-induced RNAi responses have great potential to treat a wide variety of human diseases from cancer to pandemic viral outbreaks to Parkinson's Disease. However, before siRNAs can become drugs, they must overcome a billion years of evolutionary defenses designed to keep invading RNAs on the outside cells from getting to the inside of cells. Not surprisingly, significant effort has been placed in developing a wide array of delivery technologies. Foremost of these has been the development of N-acetylgalactosamine (GalNAc) siRNA conjugates for delivery to liver. Tris-GalNAc binds to the Asialoglycoprotein receptor that is highly expressed on hepatocytes resulting in rapid endocytosis. While the exact mechanism of escape across the endosomal lipid bilayer membrane remains unknown, sufficient amounts of siRNAs enter the cytoplasm to induce robust, target selective RNAi responses in vivo. Multiple GalNAc-siRNA conjugate clinical trials, including two phase III trials, are currently underway by three biotech companies to treat a wide variety of diseases. GalNAc-siRNA conjugates are a simple solution to the siRNA delivery problem for liver hepatocytes and have shown the RNAi (and antisense oligonucleotide) field the path forward for targeting other tissue types.

Keywords: N-acetylgalactosamine; clinical trials; liver.

FIG. 1.

Delivery of GalNAc-siRNA conjugates into hepatocytes. Approximately 10⁶ ASGPR resides on the surface of liver hepatocytes, and upon binding, sialyl-GalNAc molecules are rapidly engulfed into hepatocytes by endocytosis. Due to a pH drop, GalNAc-siRNA conjugates are released from ASGPR into the lumen of the endosome, and ASGPR recycles back to the hepatocyte surface. GalNAc and the linkers are rapidly degraded off of the siRNA conjugate and by a currently unknown mechanism, a small fraction of free siRNA, likely <1%, escapes across the endosomal lipid bilayer membrane into the cytoplasm of the hepatocyte. Once in the cytoplasm, siRNAs are rapidly loaded by transactivation responsive RNA-binding protein into Ago to induce robust and sustained RNAi responses. GalNAc, N-acetylgalactosamine; ASGPR, asialoglycoprotein receptor; siRNA, short-interfering RNA.