key: cord-0880155-iiikkhhy
authors: Thangamani, Lokesh; Balasubramanian, Balamuralikrishnan; Easwaran, Murugesh; Natarajan, Jeyakumar; Pushparaj, Karthika; Meyyazhagan, Arun; Piramanayagam, Shanmughavel
title: GalNAc-siRNA conjugates: Prospective tools on the frontier of anti-viral therapeutics
date: 2021-08-30
journal: Pharmacol Res
DOI: 10.1016/j.phrs.2021.105864
sha: a022634a2782470d8d706eee954abcfb21cfeaf1
doc_id: 880155
cord_uid: iiikkhhy

The growing use of short-interfering RNA (siRNA)-based therapeutics for viral diseases reflects the most recent innovations in anti-viral vaccines and drugs. These drugs play crucial roles in the fight against many hitherto incurable diseases, the causes, pathophysiologies, and molecular processes of which remain unknown. Targeted liver drug delivery systems are in clinical trials. The receptor-mediated endocytosis approach involving the abundant asialoglycoprotein receptors (ASGPRs) on the surfaces of liver cells show great promise. We here review N-acetylgalactosamine (GalNAc)-siRNA conjugates that treat viral diseases such as hepatitis B infection, but we also mention that novel, native conjugate-based, targeted siRNA anti-viral drugs may also cure several life-threatening diseases such as hemorrhagic cystitis, multifocal leukoencephalopathy, and severe acute respiratory syndrome caused by coronaviruses and human herpes virus.

assays, and translational biology, virology, and cancer approaches exploit small (15) (16) (17) (18) (19) (20) nucleotide) non-coding RNAs that regulate the expression of entire genomes [1] . These RNAs are subdivided by their biological roles or origins; short interfering RNAs (siRNAs) and microRNAs (miRNAs) play unique and important roles in RNA interference mechanism (RNAi) pathways [2-4], activated when cells encounter a double-stranded RNA (dsRNA) that is often a sign of (possibly fatal) infection [5] . We evaluate the anti-viral efficacies reported in clinical trials of N-acetylgalactosamine (GalNAc)-based siRNA treatments for several viral diseases including Hepatitis B. It is hoped that several targeting drug delivery systems will become available in the next few years.

The RNAi mechanism was initially discovered as a form of miRNA-mediated silencing of the Caenorhabditis elegans genome [6] . A trigger RNA termed a long dsRNA or an miRNA primary transcript is cleaved and processed (by the RNase III enzymes Dicer and Drosha) into siRNAs with two-to-four-nucleotide overhangs on the 3′-ends of each strand [7, 8] (Figure 1) .

Then, the siRNAs are embedded in an effector complex termed the RNA-induced silencing complex (RISC) within which the siRNAs are matched via their stable 5′-ends, and then hybridize with the target mRNA sequence guided by the catalytic RISC protein, a member of the argonaut family (Ago2); ATP-dependent mRNA cleavage follows [9-12].

Each siRNA is associated with an Ago2-family protein to form a sequence-specific genesilencing ribonucleoprotein exhibiting specific base-pairing between the small (guide) RNA and its target mRNA sequence [13] . Recent studies on the molecular impacts of endogenous RNAi mechanisms have paved the way for use of siRNAs as nucleic acid medicines for several incurable diseases [14] [15] [16] [17] . Gene-targeting studies involving J o u r n a l P r e -p r o o f 5 novel siRNAs have reduced immune activity after organ transfer and off-target serum stability and increased siRNA potency [18, 19] ; it is possible to selectively control certain genes expressed in patients with serious genetic or viral diseases [20] . However, effective delivery of therapeutic siRNAs to target tissues remains challenging. Systemically injected nucleic acids must resist nuclease degradation in extracellular spaces, bypass renal clearance, evade sequestration by plasma proteins, avoid removal by the reticuloendothelial system, cross the capillary endothelium of the desired target cells via a paracellular or transcellular route, traverse the plasma membrane, escape the endolysosomal system prior to lysosomal degradation or reexport via exocytosis, and attain the required intracellular site of action. To date, most oligonucleotide therapeutics have focused on either local or liver delivery [21] [22] [23] [24] .

J o u r n a l P r e -p r o o f Figure 1 . RNA interference mechanism via siRNA pathway -Diagrammatic representation of the RNAi mechanism within the host cell, via externally delivered siRNA complexes designed in order to knock-down the target gene, thereby leading to gene silencing.

The virosphere is continuing to expand rapidly; new viruses are identified every year [25] [26] . The genetic-based classification of human virions is shown in Table 1 . Although viral J o u r n a l P r e -p r o o f protein structures and associations are rather well-known, the effects of the environment and host replicative properties on viral infections remain poorly understood, as do the maintenance of protein structure and function over the course of evolution [28] [29] [30] [31] [32] . This convergent evolution of gene transfer has played key roles in distributing certain protein folding patterns throughout the orders of the phylogenetic tree, establishing pathologically distinct viruses. Viral pathogens have caused successive pandemics ( 

siRNAs can be delivered to cells by viral and non-viral vectors. Synthetic siRNAs against the Influenza-A virus, incorporated in a lentiviral vector and driven by the polymerase U6 promotor, exhibited preventive and therapeutic effects when given intranasally to mice [52] [53] [54] .

A synthetic siRNA against the coxsackievirus delivered to HeLA cells via oligofectaminemediated transfection reduced viral replication [55, 56] . siRNAs developed against CoV-SARS (pSUPER and pSilent1-U6) transfected to cells with the aid of lipofectamine inhibited N gene expression; similarly, siRNA Pbs/U6 given intratracheally to mice reduced viral replication [57, 58] . A synthetic siRNA against the same RNA virus given intranasally to monkeys reduced infection and symptoms [59, 60] . Similarly, a Pcdna3/U6 siRNA against the food-and-mouth- pegaptanib, mipomersen, eteplirsen, defibrotide, nusinersen, inotersen, patisiran, and givosiran [35, 72] .

siRNA-based treatments may be very valuable, but delivery remains problematic.

Injected siRNAs may be degraded by nucleases in plasma, immune cells, and the kidney [74-78], greatly reducing the half-lives. Free siRNA does not readily cross cell membranes, given its negative charge and high molecular weight [79] . Occasionally, siRNAs may trigger non-specific side-effects. Nano-carriers and endosome-based delivery systems have been developed. Also, siRNAs have been modified via addition or removal of sugars, bases, or overhangs, and substitution of uridine residues [80] [81] [82] . GalNAc-siRNA conjugate is a trimer that binds firmly to a major hepatocellular protein, the asialoglycoprotein receptor (ASGPR) [83] . Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used to assess the chemical integrities of GalNAc-siRNA conjugates. In preclinical studies, these conjugates J o u r n a l P r e -p r o o f successfully entered the livers of both rodents and nonhuman primates and are now the subjects of several clinical trials (Table 3 ) [84] . Effects are evident when the trimer level exceeds 6 μg/mg ASGPR.

The liver constitutes one-third of the total reticuloendothelial mass of the human body, playing major roles in defense against a wide range of microorganisms [87] . Liver integrity is compromised by microbial pathogens that cause acute liver failure, hepatic fibrosis, and cirrhosis [88] . Hepatitis B is one such pathogen [89, 90] . It was earlier considered that the infection was incurable, but it can now be eradicated using nuclear-based anti-viral drugs (NUCs) including lamivudine, telbivudine, adefovir, entecavir (ETV), tenofovirdisoproxil (TDF), and tenofoviralafenamide (TAF) [91] [92] [93] [94] [95] [96] [97] . RNAi-based drugs may treat several severe viral infections (including EBOLA infections) for which effective drugs and vaccines are lacking. GalNAc-siRNA conjugates bind to the approximately 10 6 ASGPR molecules on the sinusoidal cellular membrane of a hepatocyte. The conjugates are then endocytosed and accumulate in clathrincoated pits [98] . When the pH falls, the conjugates are released into the cellular lumen and return to the cell surface [84] . Soon thereafter, the sialyl-GalNAc linkers are removed from siRNA, triggering transactivation of the RNA-binding protein and RNAi activity within cells (Figure 2 ).

The first-generation drugs (the two synthetic siRNAs of ARC-520) [99] were delivered as GalNAc conjugates to patients with chronic HBV infection; the preclinical study of Arrowhead

Pharmaceuticals is now entering the clinical phase. ARC-520 was well-tolerated in healthy volunteers (trials NCT02452528, NCT01872065, and NCT02604212) [100] . The drug targets the common regions at the 3′ UTR ends of HBV transcripts from episomal DNA; the drug is linked to a dynamic poly-conjugate (DPC) that facilitates cellular uptake by protecting it from degradation when given intravenously [101, 102] . GalNAc-siRNA conjugates are simpler, smaller, and more defined than the LNP formulations. GalNAc-siRNA is synthesized using a solid-state method and chemically defined via MS [112] . Initially a neoglycopeptide (ah-GalNAc) 3 was used to target a ligand composed of short, neutral, methyl phosphonate 8-mer oligonucleotides [113] . The linker length and sugar were then optimized, and a refined tris-galactoside structure used to deliver lipids and ASOs [114] . Recently, sequential conjugation of GalNAc residues via nucleosidic linkages has increased drug potencies [115] , enhancing hepatocyte oligonucleotide delivery to ∼10-fold that of free systems in preclinical models [116] .

Recent clinical trials using GalNAc-siRNA conjugates have been performed by Alnylam, Arrowhead, and Dicerna Pharmaceuticals. Alnylam is evaluating six GalNAc-siRNA conjugates in three phase III trials that widely target liver diseases. The first clinical trial evaluated revusiran (ALN-TTRsc) that targets the transthyretin (TTR) protein in an effort to treat TTR-mediated amyloidosis (trial nos. D2, D1-64, D1-65).

siRNAs that target key signaling genes may not only improve drug efficiencies but also enhance drug uptake and distribution by the native receptors based therapeutic approaches at cellular levels influencing chemical modifications in delivery mechanisms. We have reviewed the role played by GalNAc conjugates in oligonucleotide-based therapeutic approaches that exhibit great potential in targeted drug delivery system. Recently, ASO conjugated to 5ʹ nucleic acids has been shown to be maximally amenable to solid-phase synthesis, and to target hepatocytes more effectively, being tested against various disease conditions and more novel drugs based on native cell and tissue specific conjugates are yet to come in the future.

These challenges serve to be the major achievements of pharmaceutical industries in the upcoming era.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

This research received no external funding. J o u r n a l P r e -p r o o f

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Writing-original draft preparation

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