Oligonucleotide therapies for nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) represents a severe disease subtype of nonalcoholic fatty liver disease (NAFLD) that is thought to be highly associated with systemic metabolic abnormalities. It is characterized by a series of substantial liver damage, including hepatocellular steatosis, inflammation, and fibrosis. The end stage of NASH, in some cases, may result in cirrhosis and hepatocellular carcinoma (HCC). Nowadays a large number of investigations are actively under way to test various therapeutic strategies, including emerging oligonucleotide drugs (e.g., antisense oligonucleotide, small interfering RNA, microRNA, mimic/inhibitor RNA, and small activating RNA) that have shown high potential in treating this fatal liver disease. This article systematically reviews the pathogenesis of NASH/NAFLD, the promising druggable targets proven by current studies in chemical compounds or biological drug development, and the feasibility and limitations of oligonucleotide-based therapeutic approaches under clinical or pre-clinical studies.


INTRODUCTION
3][4][5][6] Despite obscure symptoms at a very early stage, NASH may gradually progress to cirrhosis and other end-stage liver diseases such as hepatocellular carcinoma (HCC), requiring eventual liver transplantation. 7As NASH has brought huge life-threatening concerns and economic burdens around the world, effective therapeutic approaches are urgently desired.It has been widely accepted that NASH results from numerous metabolic and pathologic alterations that proceed in parallel, including genetic predisposition, abnormal lipid metabolism, oxidative stress, lipid toxicity, mitochondrial dysfunction, inflammation, gut dysbiosis, and endoplasmic reticulum (ER) stress, 8 which certainly raise great difficulties for single-action drug development.So far, the development of chemical drugs targeting thyroid hormone receptor b (Thr-b), glucagon-like peptide 1 receptor (Glp-1R), farnesoid X receptor (Fxr), and peroxisomal proliferator-activated receptor (PPAR) are at the forefront of the drug pipelines. 9Followed by the announced positive topline results of Thr-b agonist resmetirom (MGL-3196), 10 the US Food and Drug Administration (FDA) approved resmetirom as the first-line medication for NASH patients with moderate to advanced liver fibrosis on March 14, 2024, 11 greatly boosting confidence and demands in NASH-specific drug development.
As an emerging drug-development strategy, oligonucleotide drugs have risen rapidly in recent years.Oligonucleotides refer to small DNA/RNA molecules with 8-50 nucleotides in length that bind to target RNA via Watson-Crick base pairing. 12Oligonucleotides can be normally used to inhibit gene expression through various mechanisms including RNA interference (RNAi), RNase H-mediated cleavage, and non-coding RNA (ncRNA) inhibition. 13Thanks to their potent gene-silencing capacity, oligonucleotides have been widely applied in gene therapy via both vehicle-based and vehicle-free approaches. 13,14Liver is considered an attractive organ for gene therapy due to natural hepatic tropism for many virus or non-viral vehicles. 15,16Therefore, oligonucleotide drugs are now rendering potential therapeutic options for patients with various metabolic liver diseases. 15For instance, givosiran and mipormersen are two oligonucleotide drugs approved for treating acute hepatic porphyria (AHP) 17 and homozygous familial hypercholesterolemia (HoFH), 18 which have encouraged attempts to develop oligonucleotides in the treatment of NASH.Here, we summarize the latest advances and perspectives in NASH/NAFLD pathogenesis, chemical compounds undergoing NASH-related clinical investigations, and recent innovations in liver-targeting therapeutic oligonucleotides for NAFLD/NASH.

CLINICAL PRESENTATION AND DIAGNOSIS
In 1980 NASH was, for the first time, described as a nonalcoholic disease with similar pathological features and tendency to cirrhosis as alcoholic hepatitis. 19The consensus statement published in 2022 reported that the global prevalence of NAFLD in adults was estimated to range from 23% to 25%, among whom 1 in 5 were diagnosed with NASH. 20In the United States, the number of people with NASH was predicted to reach 19.53 million by 2039. 21Nowadays, NAFLD/NASH is considered to be greatly driven by altered metabolism, whereby many metabolic factors are involved. 22To further strengthen the consensus from the field, in June 2023, the European Association for the Study of the Liver (EASL) Congress announced the new nomenclatures MASH (metabolic dysfunction-associated steatohepatitis) and MASLD (metabolic dysfunction-associated steatotic liver disease) to replace NASH and NAFLD, respectively. 23Based on 14 histological features assigned in the NAFLD activity scoring (NAS) system that was designed by the Pathology Committee of the NASH Clinical Research Network (CRN), scores reaching 5 or above correlate with increased severities of NASH diagnosis. 24Additionally, according to the practice guidance from the American Association for the Study of Liver Diseases (AASLD), patients presenting more than 5% hepatocyte steatosis and lobular inflammation (regardless of liver fibrosis) but lacking excessive alcohol consumption are diagnosed with NASH. 1 Noninvasive assessments (including "NAFLD fibrosis scoring" or "fibrosis-4 scoring," magnetic resonance elastography, ultrasound elastography, and vibration-controlled transient elastography) are usually needed for patients with comorbid conditions, persistently elevated transaminases, and/or concern for cirrhosis. 25Liver biopsy, as the only method to distinguish simple liver fatty infiltration from NASH, should be considered once inconclusive results of fibrosis are obtained from the aforementioned diagnostic methods. 25However, patients typically do not undergo liver function tests or imaging diagnosis until symptoms occur, resulting in progressive NASH conditions ahead of the time of discovery.Therefore, NASH is also known as the "silent killer."

PATHOGENESIS AND CURRENT TREATMENT APPROACHES FOR NASH
NASH differs from simple steatosis by showing more significant hepatocyte apoptosis accompanied by increased inflammation. 26The "two-hit hypothesis" suggested that NASH development requires steatosis caused by triglyceride (TG) accumulation and oxidative stress-mediated lipid peroxidation. 27Further studies held the "non-triglyceride lipotoxicity hypothesis" by elucidating that TGs played protective roles throughout NASH progression, whereas liver injury was mainly caused by non-triglyceride lipotoxic metabolites. 28In many cases, liver inflammation prior to steatosis was observed, leading to the prevailing "multiple parallel hits hypothesis" that NASH is the result of multiple factors derived especially from adipose tissue and gut. 29Overload of fatty acids (FAs) in the liver has been shown to contribute to IR and lipotoxicity [30][31][32] via disrupted mitochondria respiration 33,34 and elevated reactive oxygen species (ROS) to cause hepatocyte death. 35,36The aforementioned cellular stress could stimulate pro-inflammatory and pro-fibrogenic responses of immune cells including monocyte-derived macrophages, resident Kupffer cells (KCs), and lymphocytes, 37,38 which in turn promote extracellular matrix (ECM) production and fibrosis via activated hepatic stellate cells (HSCs). 39Moreover, toxic bile acid retention caused by disturbed hepatobiliary function has been found to be involved in NASH pathogenesis. 40,41Furthermore, recent studies have demonstrated the roles of bacterial metabolites and increased gut permeability in the progression of NAFLD/ NASH. 42,43wadays, primary treatments of NAFLD still mainly focus on lifestyle intervention.For example, limiting fructose intake is thought to improve disease conditions, as daily fructose ingestion has been shown to associate with liver fibrosis in NAFLD patients. 44In addition, aerobic exercise and adequate sleep are beneficial. 45,46Nevertheless, the efficacies of such interventions mainly rely on individuals' genetic backgrounds and/or self-discipline.Once the disease has progressed to fibrotic stages, lifestyle interventions are considered meaningless.Prior to the recent approval of resmetirom by the FDA, medications for NASH mainly aim at harnessing risk factors, including correcting dyslipidemia and hyperglycemia.For instance, vitamin E has been used in the treatment of NASH for its antioxidant properties. 47Some thiazolidinediones (TZDs), such as pioglitazone, have been shown to act as insulin sensitizers to improve metabolic status. 48,49However, the data also indicated the increased number of adverse events in pioglitazone-administered NASH patients by showing weight gain, dysregulated bone metabolism, and hemorrhagic stroke. 48,49URRENT CHEMICAL DRUG-DEVELOPING STRATEGIES FOR NASH

Targeting lipid metabolism
Dietary fat intake, plasma free fatty acid (FFA) absorption, and de novo lipogenesis (DNL) provide major sources for hepatic lipids.
Once esterified to TGs and cholesterol esters (CEs), the excessive FAs are stored in lipid droplets (LDs), where fatty acid oxidation (FAO) and very-low-density lipoprotein (VLDL) secretion are important outlets for them 50 (Figure 1).Any faulty step can render opportunities to develop liver steatosis. 51,52For instance, enhanced DNL promoted liver fat accumulation, 53 while significantly elevated FFA levels were also shown in NAFLD patients. 54In addition, the enzymatic activity of b-hydroxyacyl-coenzyme A (CoA) dehydrogenase (the rate-limiting enzyme for b-oxidation in FAO) was shown to decrease during the progression of NAFLD/NASH. 55 Based on this knowledge, Acc and Scd inhibitors were reported to ameliorate NASH by reducing steatosis, liver injury, inflammation, and fibrosis. 56,57A phase 2 study of liver-targeted Acc inhibitor firsocostat (GS-0976) has been completed (NCT02856555), showing decreased hepatic steatosis and fibrosis compared with placebo groups. 58,59Another Acc inhibitor, clesacostat (PF-05221304), was shown to possess an anti-steatosis effect in high-dose groups (NCT03248882). 60Meanwhile, the Scd inhibitor aramchol was reported to significantly alleviate liver fibrosis in NASH patients 61 and is now awaiting the phase 3 study for formulation improvement (NCT04104321). 62Fasn is another enzyme in the DNL pathway, and the misregulated expression of this factor was found to mediate proinflammatory and fibrogenic signaling. 63Recently, TVB-2640 (denifanstat), a Fasn inhibitor, finished its phase 2 trial in NASH patients (NCT04906421).On the other hand, liver FAs can also be used for TG synthesis via Dgat2 catalysis 64 (Figure 1).The phase 2 study of the Dgat2 selective inhibitor ervogastat (PF-06865571) co-administered with clesacostat in NAFLD patients has been completed with satisfactory results (NCT03776175). 60Fgf 21 and Fgf19 could serve as diagnostic markers for NASH. 65Fgf19 also regulates cholesterol 7a-hydroxylase (CYP7A1) gene transcription, which encodes the rate-limiting enzyme in bile acid synthesis. 66[72][73][74][75] Targeting insulin resistance IR results in higher insulin levels than normal because insulin-targeted tissues are less responsive in blood sugar regulation. 768][79] IR-mediated lipid metabolism disturbance may contribute to NAFLD/NASH through promotion of white adipose tissue (WAT) lipolysis and liver DNL as well as altered mitochondrial FAO. 80For example, in NAFLD patients, serum FFA levels increased due to the failure of insulin-mediated lipolysis suppression. 78Meanwhile, hyperinsulinemia and hyperglycemia in NASH patients may activate sterol-regulatory element binding protein 1c (Srebp1c) and carbohydrate response element binding protein (Chrebp), respectively, to activate DNL-related gene expression in the liver. 81Mitochondrial FA b-oxidation may increase to adapt to the upregulated lipogenesis at an earlier stage, but decompensates to such changes, eventually leading to mitochondria damage, oxidative stress, and insulin signaling impairment. 80Currently, promising therapeutic targets involved in the clinical treatments of IR-related NASH include Glp-1R, Thr-b, sodium-glucose co-transporter 1/2 (Sglt1/2), PPAR, and Fxr.
4][85][86] An FDA-approved longacting Glp-1 analog, liraglutide, was shown in a phase 2 study (NCT01237119) to improve liver function and resolve pathological manifestations in NASH individuals with or without T2D. 87,880][91][92] A phase 3 research study of single administration of semaglutide in NASH is under way (NCT04822181).However, semaglutide and liraglutide were unfortunately shown to be associated with increased risk of gastrointestinal adverse events in weight control. 93dditionally, serving as dual agonists for both Glp-1Rs and glucosedependent insulinotropic polypeptide (Gip) receptors (Gip-Rs), tirzepatide (LY3298176) and cotadutide (MEDI0382) are also undergoing clinical trials of NASH therapy (NCT04166773 and NCT04019561).Furthermore, efinopegdutide (MK-6024), the dual agonist for Glp-1Rs and glucagon receptors (Glu-Rs), has been granted a fast-track designation from the FDA recently for NASH treatment (NCT04944992).Retatrutide (LY3437943) is a triagonist of Glp-1Rs, Gip-Rs, and Glu-Rs.In recently published phase 2 results, retatrutide was demonstrated to resolve hepatic steatosis in obese patients with NASH (NCT04881760). 94The Thr-b ligand tri-iodothyronine (T3) has been shown to confer insulin-like effects by regulating functional gene expression in FA synthesis. 95The positive topline results of the Thrb-selective agonist resmetirom (MGL-3196) in the phase 3 trial (NCT03900429) were announced in December 2023. 10Very recently, it has been approved by the FDA as the first NASH-specific drug for treating patients with moderate to advanced liver fibrosis. 11The phase 2 study of another Thr-b agonist, VK2809, is under way to treat histologically confirmed NASH patients (NCT04173065).Sglt1 and Sglt2 are glucose transporters that mediate uptake through the apical cell membrane. 96Sglt1 is mainly responsible for sodium-dependent glucose uptake in the small intestine, while Sglt2 is responsible for glucose reabsorption in renal proximal convoluted tubules. 97,98Licogliflozin (LIK066), a chemical compound inhibiting both Sglt1 and Sglt2, was found to improve the liver function in obese patients with NASH in a phase 2 study (NCT03205150). 99The PPAR family members PPARa, PPAR-b/d, and PPAR-g have also been demonstrated to link with NASH via regulating lipogenesis, 100,101 FA transportation, 102 and energy utilization, [103][104][105] as well as lipotoxicity-related inflammation. 106aroglitazar has been shown to act as a PPAR-a/g agonist, decreasing liver fat content and alanine transaminase (ALT) in NAFLD/NASH patients (NCT03061721). 107Lanifibranor (IVA337), a pan-PPAR ligand that stimulates PPAR-a, -d, and -g, was reported to decrease the SAF (steatosis, activity, and fibrosis) score in patients with active NASH 108,109 and is now in a phase 3 study (NCT04849728).However, the PPAR-g-specific agonist pioglitazone was recently shown to have no increased benefit over placebo in NASH patients without diabetes (NCT00063622). 110The phase 3 study of elafibranor, which activates PPAR-a and PPAR-d, was also terminated due to low efficacy (NCT02704403). 111,112It has been shown that the bile acid receptor Fxr downregulates Cyp7a1 expression to lower bile acid level. 40,113,114xr activation was also found to inhibit the expression of Srebp1c and facilitate TG homeostasis. 115The phase 3 study (NCT02548351) of obeticholic acid (OCA), an Fxr agonist that was shown to decrease IR in NAFLD patients, 116 is now terminated.Tropifexor (LJN452) has been shown to downregulate alanine aminotransferase (AST) level and hepatic fat fraction in NASH patients, 117 but its phase 2 study was terminated (NCT02855164).Cilofexor (GS-9674), another Fxr agonist, is now in a combination therapy study with tropifexor (NCT03449446). 118rgeting hepatocyte inflammation, fibrosis, and death As mentioned earlier, increased serum FFAs and accumulated lipids in the liver could both cause liver steatosis, where lipotoxicity is considered one of the most critical mechanisms leading to the transition of NASH from NAFLD. 119Under such circumstances, hepatocyte apoptosis is induced by subsequent oxidative stress, ER stress, and other damage, [120][121][122] which in turn cause inflammation and fibrosis via activated KCs and HSCs, respectively. 123,124Hence, anti-inflammation/anti-fibrosis strategies for treating NASH are considered effective by manipulating the targets including C-C chemokine receptor type 2/5 (Ccr2/5), tumor necrosis factor (TNF-a), vascular adhesion protein 1 (Vap-1), galectin-3, and apoptosis signal-regulating kinase 1 (Ask1).
It has been shown that Ccr2-mediated hepatic infiltration of monocyte-derived macrophages (MoMFs) could directly cause inflammation and activate HSCs. 125Ccr5, another member of the Ccr family expressed on HSCs, has also been shown to promote HSC migration, proliferation, and secretion. 126,127Cenicriviroc (CVC), a dual inhibitor of Ccr2/5, had its phase 3 clinical trials in treating NASH terminated early due to lack of efficacy (NCT03028740). 128Pentoxifylline (PTX), a methylxanthine derivative attenuating the production of pro-inflammatory cytokines including TNF-a, 129 was shown to improve the histological features of NASH (NCT00590161) and is now in a phase 3 study (NCT05284448). 130Vap-1, also known as semicarbazide-sensitive amine oxidase, promotes the recruitment of pro-inflammatory cells to the liver. 131The phase 1 clinical trial of its inhibitor TERN-201 has been completed (NCT04897594).3][134][135][136] The galectin-3 inhibitor belapectin (GR-MD-02) was reported to reduce liver fibrosis in NASH patients in a phase 2 study (NCT02421094).Selonsertib (GS-4997) is a selective inhibitor targeting Ask1, a mitogen-activated protein (Map) kinase kinase kinase (Mapkkk), in response to various cytotoxic stresses. 137The therapeutic potential of selonsertib was shown in combination with firsocostat or cilofexor in a phase 2 study for treating bridging fibrosis or compensated cirrhosis due to NASH (NCT03449446 and NCT02781584). 138Notably, ER stress initiated by failed unfolded protein response (UPR) network is proven to be associated not only with metabolism disorders but also with inflammation and apoptosis. 120The AdipoR1/AdipoR2 dual agonist peptide JT003 was shown to regulate ER functions and improve liver fibrosis in mouse models. 139Another recent study has demonstrated that BGP-15, a potential poly (adenosine 5 0 -diphosphate ribose) polymerase (PARP) inhibitor, functioned in ER stress blockade and NASH mitigation when combined with olamkicept (sgp130Fc, an interleukin-6 trans-signaling blocker). 140,141

OLIGONUCLEOTIDE DRUG-DEVELOPMENT STRATEGIES FOR LIVER DISEASES
Although major obstacles including relatively lower therapeutic efficacy and tissue specificity compared with conventional chemical compounds prevent the widespread application of oligonucleotide drugs, as of December 2023 dozens of oligonucleotide drugs have received regulatory approval from the FDA.Given the high perfusion rate, discontinuous sinusoidal endothelium, and abundant receptors in the liver, oligonucleotide drugs have long been considered as the alternative approach to treat liver metabolic diseases. 142Among these approved drugs, 11 target the liver.Intensive studies in oligonucleotide therapies have shed light on treating various liver diseases, including NASH.Learning from valuable results obtained in NASH-related chemical compounds and biologics development (Figure 1), oligonucleotide drugs have been designed to target critical factors residing in, but not limited to, the aforementioned pathways.

Type of oligonucleotides and the modes of action
As small synthetic nucleic acid polymers, oligonucleotides target messenger RNA (mRNA), ncRNA, or DNA via complementary base pairing while also interacting with certain proteins through three-dimensional binding. 143Currently, antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA) mimic or inhibitor, and small activating RNA (saRNA) are the most intensively studied oligonucleotide species, with diversified action modes, including expression inhibition or activation of functional genes and non-coding transcripts as well as mRNA splicing modulation. 144

ASO
ASO is defined as a short, synthetic, single-stranded DNA, consisting of 8-50 nucleotides in length and designed to bind to RNA via Watson-Crick base pairing. 145,146Currently, ASOs make up more than 60% of oligonucleotide drugs undergoing active development. 144omivirsen is the first FDA-approved ASO drug developed for treating cytomegalovirus (CMV) retinitis. 147ASOs mainly function as expression inhibitors through the RNase H enzyme-mediated mRNA degradation pathway 146 (Figure 2).Other studies suggested that ASOs might inhibit 5 0 end capping and 3 0 end polyadenylation once bound with pre-mRNAs, leading to the destabilization of RNAs. 148Additionally, it has been reported that ASOs could be designed to bind with the intron-exon boundaries of targeted pre-mRNAs for splicing regulation. 148,149RNA siRNA refers to a 21-to 23-nt-long double-stranded RNA, usually with two free bases at the 3 0 end. 150Matured siRNAs formed by cleavage of internalized exogenous long double-stranded RNAs (dsRNAs) or short hairpin RNAs (shRNAs), have been demonstrated to introduce cleavage or degradation on mRNA targets. 151Artificially designed siRNAs with perfect base-pair matching can be synthesized and transfected into host cells for gene transcription manipulation.
In the cytosol, siRNA duplexes participate in the formation of RNA-induced silencing complex (RISC) with Argonaute 2 protein (Ago2), resulting in separated single strands. 152Once the RISCbound antisense sequences specifically match the target mRNAs, mRNA cleavage is induced by Ago2, followed by RNase-mediated hydrolysis 153 (Figure 2).Notably, because RISC-bound siRNAs are protected from nuclease degradation, they can render prolonged effects via siRNA recycling and repeated degradation of mRNAs. 154RNA mimic or inhibitor miRNAs were primarily discovered as endogenous ncRNAs involved in RNA-mediated gene silencing in mammalian cells.155 RNA polymerase II mediates miRNA transcription in the nucleus by forming primary miRNA (pri-miRNA) transcripts.156 These transcripts are cleaved by Drosha and co-factor protein Dgcr8, resulting in precursor miRNAs, namely pre-miRNA.157 Once translocated to the cytoplasm and further cleaved by Dicer along with transactivation-responsive RNA-binding protein (Trbp) to form miRNA duplex, one strand of miRNA binds with RISC, leading to translational inhibition or degradation on target mRNA.158,159 Due to their ability to manipulate mRNA abundance, synthetic miRNA mimics or inhibitors have been developed as applicable therapeutic approaches for various diseases.miRNA mimics are synthetic RNA duplexes containing strands identical to those of the corresponding miRNAs, facilitating the restoration or enhancement of miRNA functions.160 On the other hand, inhibiting miRNA function can be achieved by using anti-miRNA oligonucleotides (anti-miRs). 161Anti-miRs are single-stranded oligonucleotides structurally similar to ASOs, which have been shown to directly bind with the target miRNAs, displaying promising utilizations in miRNA therapeutics.161 Currently, phase 2 trials of miRNA mimics for keloid treatment (NCT03601052) 162 and anti-miRs, known as miravirsen, for hepatitis C virus (HCV) therapy (NCT01200420) have been completed.163 saRNA Unlike the gene-silencing oligonucleotides mentioned above, saRNAs are 21-nt double-stranded RNAs that interact with promoters to induce transcriptional activation in an Ago2-dependent manner.167 For instance, hepatocyte nuclear factor 4a (Hnf4a) is a crucial liver-specific transcription factor to mediate hepatocyte differentiation, 168 liver morphogenesis, 169 and lipid metabolism.170 Liver-specific deletion of HNF4a in mice displayed deleterious effects in increasing liver lipid accumulation.171 Huang et al. developed saRNA oligo-dendrimers targeting HNF4A P1 promoter to enhance HNF4A expression.The results showed favorable metabolic profile change with reduced liver TGs and IR improvement in high-fat diet (HFD)-fed rats, indicating that saRNA-mediated HNF4A activation may represent a new therapeutic strategy for NAFLD and IR. 172

Modifications of synthetic oligonucleotides
To improve specific and effective delivery to target tissues, chemical modifications of synthetic oligonucleotides have been proven as necessary strategies.These modification strategies can be applied to nucleic acid backbone, ribose sugar, and nucleobase singly or in combination to enhance the stability and efficacy of oligonucleotide drugs. 13In particular, the modifications on oligonucleotide backbones have involved primarily replacing phosphodiester (PO) linkages with phosphorothioate (PS) linkages.In this process, sulfur atoms are utilized to substitute non-bridging oxygen atoms of the internucleotide phosphate group to increase nuclease resistance. 173alancing the ratio between PO and PS linkages residing in the same oligonucleotide molecule is considered critical to reducing undesired effects such as prolonged retention and compromised target binding. 174Notably, Rp and Sp isomers are two configurations for PS linkages.It has been shown that PS linkages with the Sp configuration are more stable than its stereochemical counterparts. 175A study team from Wave Life Sciences demonstrated that the DNA region with an (RpSpSp) 3 core within ASO Gapmer (described below) were more effective than a stereorandom arrangement in leading RNase H1-mediated degradation on target mRNAs. 175Moreover, 5 0 -phosphate terminal modifications were developed to enhance the efficacies of siRNAs, as the phosphorylated 5 0 end of the guide strand was found to interact with the middle domain of Ago proteins. 176The newly developed 5 0 -phosphate analogs including 5ʹ-C-methyl, 5ʹmethylenephosphonate, and 5ʹ-vinylphosphonate are shown to have conformations and steroidal electronic properties similar to those of natural phosphates while displaying resistance against dephosphorylases. 177,178Ribose sugar modifications are commonly designed to substitute the 2ʹ-hydroxyl group on RNA with 2ʹ-O-methyl (2ʹ -OMe), 2ʹ-O-methoxyethyl (2ʹ-MOE), or 2ʹ-fluoro (2ʹ-F), which have been verified to increase the half-lives of oligonucleotides in plasma and improve their binding affinities [179][180][181][182] but cannot lead to RNase H activation. 183,184 Bridged nucleic acids (BNAs) are featured by a linkage joining the 2ʹ oxygen to 4ʹ carbon between the ribose, 185 including locked nucleic acid (LNA), 186 2ʹ,4ʹ-constrained 2ʹ-O-ethyl (constrained ethyl) BNA (cEt), 187 and 2ʹ-O,4ʹ-Cethylene-bridged nucleic acid (ENA). 188The most commonly used LNA has been found to significantly improve the thermodynamic stability and nucleic acid recognition potential with increased melting temperature. 189Further studies have developed alternative chemis-tries to alter the original DNA or RNA structures, resulting in excellent resistance against various enzymes and unwanted aggregation once linked with charged bioconjugates, such as cationic cell-penetrating peptides (CPPs, described in "other bioconjugations"). 190,191or instance, peptide nucleic acids (PNAs) have aminoethylglycine backbones with acetyl linkers, 192 while phosphorodiamidate morpholino oligomers (PMOs) have backbones consisting of morpholine rings that bear methylene groups. 193In addition, unlocked nucleic acids (UNAs) with unconnected 2ʹ and 3ʹ carbons, 194 glycol nucleic acids (GNAs) using propylene glycol to alter ribose or deoxyribose, 195 and tricyclo-DNAs (tcDNAs) with an additional ethylene bridge between the 3ʹ and 5ʹ carbons have also been tested. 196Strategies for nucleobase modifications have also been widely investigated.For instance, 2-thiouridine, pseudouridine (Psi), and dihydrouridine have been shown to enhance the thermodynamic stability and gene-silencing efficacy of particular siRNAs/ASOs. 197

ASO
The earliest attempts at ASO modification mainly included PS linkage, leading to the advent of FDA-approved fomivirsen 198 (Figure 3).However, studies have shown that PS may cause compromised interaction between ASO and target mRNA. 199As the second www.moleculartherapy.orgReview generation of ASOs, Gapmer is a short central DNA segment flanked by RNA-based sequences on both sides. 13Due to the hybrid structure that is resistant to nuclease and allows modifications on the RNA flanks, Gapmer has been shown to display improved target-binding ability. 200,201Inotersen, utilizing the Gapmer structure to target transthyretin (TTR) mRNA for the treatment of hereditary transthyretin (hATTR)-mediated amyloidosis, was successfully developed and approved by the FDA in 2018 202 (Figure 3).More advanced strategies such as LNA, PNA, and PMO are prevalently adopted in recent ASO design. 203Eteplirsen and golodirsen, utilizing PMO technology, were approved by the FDA in 2016 and 2019, respectively, for the treatment of Duchenne muscular dystrophy (DMD) 204,205 (Figure 3).

siRNA
The aforementioned PS backbone and ribose sugar-modification strategies [180][181][182] have also been widely used in innovating siRNA drugs. 206For example, patisiran is a 2ʹ-OMe modified siRNA-based drug approved by the FDA in 2018 to silence mutated TTR expression in hATTR liver 207,208 (Figure 3).0][211] The FDA-approved anti-ALAS1 RNAi drug givosiran is an example that introduces both 2ʹ-OMe and 2ʹ-F modifications 208 (Figure 3).Special modification patterns were developed by Alnylam Pharmaceuticals, including standard template chemistry (STC), enhanced stabilization chemistry (ESC), advanced ESC, and ESC-Plus (ESC+).STC pattern was designed as an alternative, with 2ʹ-OMe and 2ʹ-F modifications employed in both siRNA strands except three consecutive 2ʹ-F modifications placed at positions 9, 10, and 11 of the passenger strand and consecutive 2ʹ-OMe modifications placed at positions 11, 12, and 13 of the guide strand. 206dditionally, two PS linkages are added at the 3ʹ end of the guide strand.Although the new pattern invested siRNAs with higher stability and affinity, safety remains a significant concern. 212To reduce toxicity and further improve stability, fewer 2ʹ-F modifications and four more PS linkages were added to the ESC pattern. 213Givosiran, lumasiran, and vutrisiran are successful representatives of the ESC pattern 17 (Figure 3).Thereafter, Alnylam explored multiple modification design variants by changing the proportion and position of 2ʹ-F and 2ʹ-OMe. 206ompared to the former ESC pattern, ESC+ introduced a GNA at position 7 of the guide strand, which was shown to reduce the off-target effects of N-acetylgalactosamine (GalNAc) siRNAs. 214,215Now the ESC+ pattern is applied to the development of new drugs, as seen in ALN-HBV02 for treating chronic hepatitis B virus (HBV) infection (NCT03672188) and zilebesiran for treating hypertension (NCT05103332).

miRNA mimic or inhibitor
For miRNA-based therapy, modifications including PS, LNA, and 2ʹ-OMe are widely utilized to protect oligonucleotides from RNase-mediated degradation.Because the guide strand in miRNA mimics needs to be recognized by RISC, fewer modifications (such as 2'-F modification) are available, while the passenger strand can be modified (such as 2ʹ-OMe) and linked to bioconjugations (such as cholesterol). 2168][219][220][221][222][223] Currently, miravirsen, an anti-miR-122 modified with LNA, was developed by Santaris Pharma for chronic HCV genotype 1 infection treatment 224 (Figure 3).Studies have shown that by adding LNA modification, anti-miRs significantly antagonized the endogenous miRNAs. 225,226In addition, a variety of different sequences are designed as double-stranded domains or hairpin structures and added on both ends of anti-miR to improve the binding affinity and nuclease stability. 227Furthermore, Krützfeldt et al. innovated a special modification combination "antagomir" by using 2 0 -OMe sugar modification, PS backbone modification, and cholesterol conjugation on the 3 0 end. 228hanks to the specific, efficient, and long-lasting gene silencing, antagomir is now widely used in in vivo tests. 229,230fety issues of synthetic oligonucleotides The common adverse drug reactions (ADRs) of oligonucleotides reported in various clinical studies include injection-site reactions, headache, fever, and hypersensitivity, 231 making oligonucleotidemediated side effects a big concern.
Mechanistically, by base pairing with targeted mRNA sequences, oligonucleotides may cause on-target or off-target toxicities. 232On-target toxicities refer to exaggerated intended effect (e.g., too strong silencing of the targeted mRNA) and/or target-gene silencing in unwanted organs. 232To avoid such problems, tissue-specific delivery systems are needed, while accurate assessments of tissue-related expression pattern and biological function in disease-relevant cell lines or primary human cells should be conducted in pre-clinical investigations. 233On the other hand, off-target toxicities are adverse pharmacological effects caused by undesired silencing on unrelated transcripts. 232In terms of this issue, in silico screening and in vitro/in vivo targeting evaluation are widely used, 234,235 while transcriptomics analysis to evaluate hybridization specificity is also suggested. 236her toxicities independent of base pairing can cause inflammation responses, impaired coagulation, and abnormal complement activation, as well as tissue damage in kidney and liver. 232For example, most of the earlier generation of siRNA drugs, such as genasense for the treatment of melanoma, were shown to trigger unmethylated cytosine phosphate-guanine (CpG) motif-induced immune stimulation. 237,238ASO-based ISIS2302 targeting intercellular adhesion molecule 1 (ICAM-1) was found to inhibit coagulation in cynomolgus monkeys. 239To solve these issues, precise determinations of safe concentration and efficiency of oligonucleotides are imperative.Moreover, introducing novel chemical modifications (such as 2ʹ-hydroxyl group substitution and PMO) is currently being tested and applied.Intriguingly, several modification species aiming to increase binding affinity to mRNAs, such as LNAs, may also bring extra risks to offtarget toxicities. 240,241Therefore, it is crucial to find the proper kinetics between oligonucleotide drug and its pharmacological target in the particular disease condition.

Hepatic delivery systems of oligonucleotides
The liver is the largest visceral organ in the body, with a unique circulatory system 142 where a great number of metabolic targets are susceptible to be regulated by various therapeutic nucleic acids, including oligonucleotides. 15To develop effective delivery methods for liver-targeting oligonucleotides in clinical applications, intensive studies have utilized various approaches including chemical modifications, GalNAc conjugates, liposomes, and viral vectors. 15To date, the GalNAc-conjugate platform has been proven to be an accessible solution for hepatocyte-targeted oligonucleotides. 242Based on the sophisticated chemical modification technologies (STC, ESC, advanced ESC, and ESC+) in combination with GalNAc, Alnylam Pharmaceuticals has innovated a series of FDA-approved RNAi drugs (givosiran, lumasiran, and vutrisiran) and several oligonucleotide candidates currently undergoing clinical trials.On the other hand, lipid nanoparticles (LNPs) could achieve hepatocyte-specific delivery via apolipoprotein E (ApoE)/low-density lipoprotein receptor (LDLR) interaction. 243,244Despite the high transduction efficiency, virus-based delivery approaches are mainly used to demonstrate proof of concept for the therapeutic potential of certain oligonucleotides because of safety concerns. 245Collectively, liver-targeting oligonucleotide delivery platforms are becoming more mature and implementable, laying the foundation for the development of oligonucleotide drugs to treat NASH.

GalNAc conjugates
The asialoglycoprotein receptor (ASGPR) was discovered as a lectin in rabbits by Gilbert Ashwell and Anatol Morell in 1965. 2464][255] It then dissociates from ASGPR upon endosome lumen pH drop, resulting in degradation of GalNAc and membrane recycling of ASGPR. 256,257Rogers and Kornfeld initiated liver-targeted cargo delivery via ASGPR by transferring fetuin glycopeptide-coupled proteins into the rat liver. 258Subsequently, researchers sought to deliver different substances into hepatocytes through this pathway, including therapeutic glycolipids, 259 chemotherapy drugs, 260 and nucleotides. 261,262Hangeland et al. achieved the successful delivery of an oligodeoxynucleoside methylphosphonate neoglycopeptide conjugate, [YEE (ah-GalNAc) 3]-SMCC-AET-pUmpT7, into human hepatocellular carcinoma cells (HepG2) in 1995. 2635][266] Prakash et al. developed a triantennary GalNAc-conjugated ASO, improving the potency of hepatocyte-targeted delivery by 10-fold in mice. 267Notably, GalNAc conjugated with ASOs or siRNAs now are shedding light on the clinical applications of liver-targeted oligonucleotide drugs.For instance, givosiran was designed to utilize the ESC-GalNAc delivery platform targeting ALAS1, a key enzyme gene upregulated in AHP. 17 Lumasiran and vutrisiran were designed for liver-targeted gene silencing of hydroxyacid oxidase 1 (HAO1) in primary hyperoxaluria type 1 (PH1), 268 and TTR in hATTR amyloidosis, 269 respectively (Figure 3).Meanwhile, Ionis Pharmaceuticals is leading the ongoing ligand-conjugated ASO (LICA) program, which began with the GalNAc conjugation platform developed to achieve liver-targeted inhibition of TTR mRNA and apolipoprotein C3 (APOC3) mRNA. 144,270,271her bioconjugations In addition to GalNAc, other bioconjugations, including lipids, peptides, aptamers, and antibodies, have also been tested.Cholesterol and its derivatives, linked with the 3 0 ends of passenger stands, are considered some of the most attractive lipid conjugates.Cholesterol-conjugated siRNAs have been shown to exhibit stronger binding to lipoproteins to enhance cellular transportation and uptake. 272Moreover, long-chain FAs and a-tocopherol are used to enhance siRNA delivery efficiencies to the liver. 272,273Peptide conjugates, such as CPPs, which are short cationic and/or amphipathic peptides typically equipped with fewer than 30 amino acids, have demonstrated the ability to cargo different molecules and traverse biological membranes via peptide-mediated uptake mechanisms. 274Therefore, CPPs are usually introduced to enhance the bioavailability and the target tissue uptake of oligonucleotides. 275Aptamers and antibodies are potentially optimal conjugates for delivering oligonucleotides into other cells and tissues due to their specific interactions with non-hepatocyte surface receptors. 276,277pid nanoparticles LNPs, utilizing physiologically relevant lipids as nanocarriers, are considered low in toxicity and biocompatible. 278It has been demonstrated that LNPs can be internalized via the endocytosis process followed by endosomal escape to facilitate the release of oligonucleotides in the cytosol. 279LNPs typically consist of four lipid components: distearoylphosphatidylcholine (DSPC), cholesterol, ionizable cationic lipid, and polyethylene glycol (PEG)-lipid.DSPC and cholesterol are related to LNP structure formation. 280Ionizable cationic lipids are used to improve membrane fusion efficiencies and avoid immune responses via low surface charge at physiological pH, 281 while PEG-lipids are added to control particle size and prevent aggregation. 282,283LNPencapsulated siRNA cargoes have been shown to accumulate in hepatocytes, KCs, and sinusoids, while the strongest gene-silencing effect is typically achieved in hepatocytes. 284LNPs can be further modified to enhance binding specificities toward hepatocytes 244 and HSCs 285 by conjugating with GalNAc and vitamin A, respectively.
Intensive studies have shown the therapeutic potential of LNP-encapsulated oligonucleotides delivered to the liver for treating various diseases. 282For instance, the aforementioned patisiran is an approved LNP-RNAi drug 207 that utilizes the ionizable cationic lipid dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and results in more than two orders of silencing effect compared to the original 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA). 286The PEG-lipid in this system is the shorter dimyristyl (C14) chain, which has been shown to mitigate the negative impacts of PEG shielding on siRNA silencing in vivo. 287Moreover, clinical trials are under way for the LNP-encapsulated siRNA ARB-001467 for treating HBV infection (NCT02631096) and BMS-986263 for treating liver fibrosis (NCT03420768). 288Notably, LNP-encapsulated siRNAs targeting highmobility group box 1 (HMGB1) 289 and methylation-controlled J protein (MCJ) 290 have been tested in pre-clinical NASH models, respectively.In addition, LNPs have demonstrated the ability to deliver miRNA into the liver.For instance, an miR-30a-5p mimic was encapsulated into lipidprotamine-hyaluronic acid (LPH) nanoparticle modified with HSC-targeting aminoethyl anisamide (AEAA) to treat liver fibrosis in mice. 291

Viral vectors as proof-of-concept research approaches
Since 1990, when retrovirus was first applied for clinical gene therapy of adenosine deaminase (ADA)-deficient severe combined immunodeficiency (ADA-SCID), 292 viral vectors for the delivery of nucleotide agents have rapidly developed.Lentiviruses (LVs), adenoviruses (AdVs), and adeno-associated viruses (AAVs) are three major types of viral vehicles currently used. 293Due to relatively lower relevance to human diseases, compromised immunogenicity, and cytotoxicity, AAVs are nowadays considered safer viral vectors for in vivo expression of oligonucleotide molecules. 294In addition, tissue tropism varies greatly in different AAV serotypes, 295 among which AAV8 has been shown to be a reliable vector to transduce for hepatocytes. 296Therefore, despite the controversies on AAVs as a suitable system for NASH therapy, this delivery platform has been intensively utilized in therapeutic target discovery.By introducing shRNA or pri-miRNA expressing cassettes that are driven by hepatocyte-specific promoters into viral vectors, AAVs can be used as a potent liver-targeted delivery approach for mRNA-modulating regions (e.g., siRNAs, miRNAs, and anti-miRNAs).For example, AAV-anti-miR-20b was shown to slow NAFLD progression by upregulating FAO and attenuating IR. 297 AAV6-mediated in vivo expression of the shRNA against pyruvate kinase L/R (PKLR) was reported to lower L-type pyruvate kinase expression in the liver of mice fed a high-fat and sucrose (HF/HS) diet, leading to alleviated IR and reduced liver steatosis. 298AAV8 harboring shRNA against SMS1 (sphingomyelin synthase 1) was administered in mice fed a high-fat/cholesterol diet (HFHCD), resulting in lowered expression of pro-inflammatory factors and collagen type III a1. 299

CURRENT STATUS OF RESEARCH IN OLIGONUCLEOTIDE DRUG DEVELOPMENTS FOR NASH
To date, various chemical compounds or small peptides have been developed to modulate a large number of potential therapeutic targets for NASH.Most of these target proteins mainly serve as enzymes or ligands/receptors, leaving insufficient pharmacological approaches applicable for other "less druggable" targets.Alternatively, emerging oligonucleotides are expected to modulate these targets through transcriptional regulation, offering new hopes for NASH treatments (Figure 4).In this context, we have summarized oligonucleotide therapeutics in NASH clinical trials and major pre-clinical studies (Tables 2 and 3).

Patatin-like phospholipase domain-containing protein 3
Patatin-like phospholipase domain-containing 3 (PNPLA3) encodes Pnpla3 protein with TG hydrolase activity in hepatocytes. 300Amino acid substitution from isoleucine (I) to methionine (M) at position 148 (I148M) has been reported to have a robust association with various liver metabolic diseases including steatosis and fibrosis/cirrhosis, 300 probably due to reduced enzymatic activity. 301Further studies have shown that ubiquitylation and proteasome-mediated Pnpla3 degradation were impaired by the I148M substitution, leading to the accumulation of mutated Pnpla3 in LDs and enhancing steatosis. 302,303Moreover, the overexpression of Pnpla3 I148M in an NAFLD mouse model upregulated the transcription of several marker genes involved in UPR and induced the accumulation of oxidized glutathione, suggesting its association with ER and oxidative stress. 304In a pre-clinical study, S-cEt-modified 16-mer ASOs were screened for optimal targeting on the mouse PNPLA3 gene. 305The resultant ASO was further modified by 5 0 end conjugation with triantennary GalNAc. 305The potency of anti-PNPLA3 ASO-GalNAc in improving NAFLD conditions caused by mutated PNPLA3, including liver fibrosis, was proven. 305Furthermore, ASO/ASO-GalNAc conjugate AZD2693(ION839) was innovated by Ionis Pharmaceuticals and AstraZeneca to inhibit PNPLA3 expression. 306A phase 2 study of AZD2693 with NASH patients carrying Pnpla3 I148M has been launched (NCT05809934).

Diacylglycerol acyltransferase 2
Diacylglycerol acyltransferase 2 (Dgat2) catalyzes TG synthesis from diacylglycerol and fatty acyl CoA as substrates. 64DGAT2-knockout mice were found dead soon after birth due to lipopenic phenotypes, such as dysregulated energy metabolism and impaired skin barrier function. 307Given that TG accumulation is considered one of the key steps in NAFLD pathogenesis, 27 ASO-mediated DGAT2 silencing was developed.Results showed that ASOs targeting DGAT2 significantly reduced hepatic lipid storage in rats, accompanied by lowered expressions of lipogenic genes (SREBP1c, ACC1, SCD1, and mtGPAT) and elevated expressions of oxidative/thermogenic genes (CPT1 and UCP2). 308A parallel study showed administrations of ASOs targeting DGAT2 in HFD-fed mice and ob/ob mice both efficiently reduced liver Dgat2, resulting in lowered intrahepatic TG level and attenuated hyperlipidemia, as well as reduction of hepatic steatosis. 309However, further studies using the NASH mouse model induced by a diet deficient in methionine and choline (MCD) showed that ASO-mediated DGAT2 silencing aggravated hepatic inflammation and fibrosis via elevated FFA-associated oxidative stress, 310 indicating critical roles of non-TG lipid products initiating hepatotoxicity in NASH progression.ION224 is a DGAT2-targeting ASO innovated by Ionis Pharmaceuticals. 311The phase 2 clinical trial of ION224 (NCT04932512) was completed with positive results showing improvement in NAS score without worsening fibrosis in NASH patients. 312her targets in lipid metabolism LD-associated protein serine/threonine protein kinase 25 (Stk25) has been demonstrated to play an inhibitory role in regulating lipid oxidation and insulin sensitivity. 313Biopsy data showed a positive correlation between Stk25 abundance and fat content in human livers. 314In addition, STK25 transgenic mice displayed a dramatic increase in liver lipid deposition, hepatic IR, and steatohepatitis. 314Consistently, repressed NASH symptoms including liver steatosis and oxidative damage were found in STK25-knockout mice. 315Cansby et al. designed a triantennary GalNAc-conjugated ASO for hepatocyte-targeted STK25 silencing (GalNAc-STK25 ASO), which displayed alleviated NASH symptoms in mice under chronic exposure of dietary lipids without obvious systemic toxicity or local tolerability concerns. 316Currently, Sprint Bioscience and Gothenburg University are conducting GalNAc-STK25 ASO for NASH and T2D treatment in humans.
Mammalian sterile 20-like 3 (Mst3, also known as Stk24) is another LD-associated protein closely related to Stk25. 317Chemical modified ASOs targeting MST3 have also shown the capacity to ameliorate diet-induced NAFLD, including the reduced oxidative stress and ER stress biomarkers (4-hydroxynonenal, 8-oxoguanine, KDEL, and CHOP) in mouse livers. 318hesion G-protein-coupled receptor F1 (Adgrf1) belongs to the G-protein-coupled receptor (GPCR) family. 319Recent studies found that Adgrf1 acted as an upstream regulator of Scd1. 320Moreover, two GalNAc-conjugated ASO-ADGRF1s that bind to different regions of ADGRF1 mRNA have been found to improve glucose ho-meostasis, alleviating lipid abundance and liver damage in HFD-fed ADGRF1-overexpressed mice. 320oprotein convertase subtilisin/kexin type 7 (PCSK7) encodes Pcsk7 as a transmembrane protease, 321 whose single-nucleotide variation (rs236918) is linked with dyslipidemia and liver damage in NAFLD patients. 322The recent studies led by Sachan et al. have shown that GalNAc-ASO selected to target PCSK7 mRNA had the ability to accelerate the recovery of high-fat/fructose/cholesterol (HFFC) dietinduced mice exhibiting hepatic steatosis. 323ll-like receptor 9 By serving as pattern-recognition receptors (PRRs), Toll-like receptors (TLRs) were demonstrated to recognize unwanted or mislocated DNA fragments, such as unmethylated CpG-DNA motifs from bacteria or virus genome, to initiate tissue inflammation. 324Meanwhile, a substantial amount of mitochondrial DNA (mtDNA) was also found in the plasma of NASH patients as well as HFD-fed mice, 325 where mtDNA was shown to be released into extracellular milieu from Drugs are categorized according to their targets in the NASH pathogenesis.TLR9, Toll-like receptor 9; HSP47, 47-kDa heat-shock protein; STK25, serine/threonine protein kinase 25; HMGB1, high-mobility group box 1; TAZ, transcriptional co-activator with PDZ-binding motif; HSD17B13, 17b-hydroxysteroid dehydrogenase 13; DGAT2, diacylglycerol acyltransferase 2; PNPLA3, patatin-like phospholipase domain-containing 3; MCJ, methylation-controlled J protein; SirT1, silent information regulator 1; RTK, receptor tyrosine kinase; FAS, fatty acid synthase; AEG-1, astrocyte elevated gene 1; SREBP-1c, sterol-regulatory element binding protein 1c; CHREBP, carbohydrate response element binding protein; CYP7A1, cholesterol 7a-hydroxylase; PPAR, peroxisomal proliferator-activated receptor; Ihh, Indian hedgehog; WAT, white adipose tissue; FFA, free fatty acid; DNL, de novo lipogenesis; TG, triglyceride; CE, cholesteryl ester; LD, lipid droplet; VLDL, very-low-density lipoproteins; FAO, fatty acid oxidation; mito, mitochondria; ROS, reactive oxygen species; HSC, hepatic stellate cell.www.moleculartherapy.orgReview injured hepatocytes. 326In line with these findings, the mRNA level of TLR9 (a member of the TLR family) was reported to increase in the livers of NASH patients and atherogenic diet-fed mouse models. 327oreover, pro-inflammatory cytokines in the liver were demonstrated to be mediated by activated Tlr9 along within NASH progression, where Tlr9 antagonist IRS954 could block this process. 325TLR9 knockout led to less liver steatosis, fibrosis, and IR in mice fed with choline-deficient/amino acid-defined (CDAA) diet, probably due to suppressed interleukin-1b and nuclear factor kB (NF-kB) signaling. 328All of the above data strongly suggested critical roles of Tlr9 in NASH progression.AVO101, a phase 2-ready TLR9 ASO, was developed by Shepard et al. to display elevated adiponectin, lowered weight, and reduced NASH symptoms in a primate obesity model. 329

Notch signaling pathway
The Notch signaling pathway is a conserved cellular process well known to be involved in organ formation and morphogenesis. 330Under physiological conditions, the Notch pathway was found to be required for bile duct development in nonparenchymal cells but inactive in hepatocytes. 331Interestingly, positive correlations between Notch activity in hepatocytes and NASH progression were observed in patients and diet-induced mouse NASH models. 332In addition, forced Notch activation was shown to promote secretion of the fibrogenic factor osteopontin (Opn), leading to the activation of HSCmediated fibrosis. 332g-Secretase is an enzyme catalyzing Notch intramembrane proteolysis to facilitate downstream reactions. 333herefore, various approaches to inhibit g-secretase have been considered to treat NASH.Given that the commonly used g-secretase inhibitor (GSI) was found to cause goblet cell metaplasia, 334 a liver-selective ASO to target NCST (the gene encoding one of the g-secretase complex subunits for ligand-dependent Notch activation) was developed. 332Results showed suppressed HSC activation and collagen deposition along with lowered body weight and adiposity in mice. 332Moreover, the absence of intestinal toxicity during NCST ASO administration indicated its safety via specific targeting of the inappropriately activated Notch signaling in hepatocytes. 332

Long non-coding RNAs
The essential roles of ncRNAs in NAFLD/NASH pathogenesis has been elucidated in recent studies. 335Long-ncRNAs (lncRNAs) are large ncRNA transcripts (longer than 200 nt), which are involved in post-transcriptional regulation by directly interacting with proteins or sponging miRNAs (protecting target mRNAs from miRNA binding and degradation). 336,337 a multi-functional lncRNA, nuclear paraspeckle assembly transcript 1 (NEAT1) has been demonstrated as a therapeutic target in several disease conditions.For instance, ASO-based NEAT1 silencing has been utilized in preventing post-stroke LD agglomeration. 338Since the level of this lncRNA was found to upregulate in NAFLD and liver fibrosis patients, 339,340 silencing NEAT1 by shRNA or siRNA was shown to suppress liver fibrosis and inflammation probably through disrupting the binding with miR-122 and miR-506. 340,341Other studies reported reduced lipid accumulation by shRNA-mediated NEAT1 silencing by derepressing miR-146a-5p and miR-212-5p. 339,342These results indicated that NEAT1 is a promising lncRNA target for ASObased NAFLD treatment through multi-target regulation.PNPLA3, patatin-like phospholipase domain-containing 3; DGAT2, diacylglycerol acyltransferase 2; HSD17B13, 17b-hydroxysteroid dehydrogenase 13; HSP47, 47-kDa heat-shock protein.
Given that NASH is highly related to metabolic disorders including IR, diabetes, and diabetic complications, ASO-mediated treatments targeting metabolism-regulatory lncRNAs are thought to confer potential benefits to treat this syndrome. 343AstraZeneca developed a Glp-1-conjugated ASO targeting lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), to achieve pancreatic b cell-specific oligonucleotide uptake for treating diabetes, [344][345][346] whose application could be transferred to improve dysregulated liver metabolism in NASH.Like PNPLA3 and STK25, 17b-hydroxysteroid dehydrogenase 13 (HSD17B13) also encodes an LD-associated protein mainly expressed in hepatocytes. 347Both the protein and mRNA levels of this gene were observed to be upregulated in human NAFLD liver samples. 348,349dditionally, individuals carrying HSD17B13 loss-of-function variant (rs72613567: T/A) were found to have reduced risks of NASH and cirrhosis. 350,351Furthermore, AdV-mediated overexpression of human HSD17B13 led to a fatty liver phenotype in mice, 348 highlighting its role in promoting NAFLD/NASH pathogenesis.ALN-HSD, a GalNAc-conjugated siRNA, was developed by ESC+ GalNAc-conjugate technology to silence HSD17B13 expression. 352A phase 2 clinical study of subcutaneously administered ALN-HSD for NASH therapy (NCT05519475) is currently led by Alnylam Pharmaceuticals.Meanwhile, ARO-HSD (GSK4532990) siRNA developed by Arrowhead Pharmaceuticals has completed the phase 1 clinical trial (NCT04202354), showing good tolerance with lowered hepatic HSD17B13 expression as well as decreased serum ALT level in NASH patients. 353In addition, Ionis Pharmaceuticals and AstraZeneca developed ION455 (AZD7503) based on LICA ASO targeting HSD17B13 and is currently launching phase 1 studies (NCT05143905 and NCT05560607). 354

PNPLA3
As mentioned earlier, the PNPLA3 I148M variant has been demonstrated as one of the key factors causing hepatocyte lipid accumulation. 303AMG 609 is essentially an siRNA that selectively targets the mutated allele.A phase 1 clinical trial evaluating the safety, tolerance, and liver fat changes upon subcutaneously administered AMG 609 has been launched (NCT04857606).Meanwhile, other siRNA drug candidates, JNJ-75220795 (ARO-PNPLA3) 363 and ALN-PNP, designed for reducing PNPLA3 expression, are also undergoing phase 1 trials for NASH treatment (NCT04844450, NCT05039710, and NCT05648214).
Methylation-controlled J protein MCJ (also called DnaJC1), located in the mitochondrial inner membrane, was identified as a co-chaperone to inhibit the functions of electron transfer chain (ETC) complex I. 364 As the ETC serves as the outlet for products of FA b-oxidation, an excessive amount of MCJ may contribute to NAFLD development via abnormally increased FA accumulation. 52In fact increased MCJ expression has been reported in NAFLD patients, while reduction of liver steatosis and fibrosis were observed in MCJ-deficient mouse NASH models. 2905][366][367] Since the increase in ROS production from hyperactivated ETC normally impairs mitochondria and aggravates the tissue damage, it is believed that reduction of MCJ expression might be a feasible strategy to prevent NAFLD progression. 368LNP-siRNA targeting MCJ (LNP-siMCJ) was shown to result in reduced lipid accumulation, fibrosis, and hepatocyte damage in several NASH models mimicking different disease conditions. 290alNAc-siRNA targeting MCJ (GalNAc-siMCJ) was also tested to achieve comparable therapeutic effects.290 Astrocyte elevated gene 1 Previous studies have shown the stimulatory roles of astrocyte elevated gene 1 (AEG-1) in the NF-kB pathway to induce inflammation in hepatocytes and macrophages. 369Srivastava et al. showed that Aeg-1 protein levels were significantly overexpressed in biopsy samples from NASH patients. 370In addition, spontaneous NASH-related pathological changes were observed in transgenic mice with hepatocyte-specific overexpression of AEG-1, whereas hepatocyte-specific AEG-1 knockout was shown to protect mice from HFD-induced NASH. 370The versatile functions of Aeg-1 in promoting NASH may be attributed to enhanced DNL and inflammation as well as downregulated FAO in the liver. 370Previously validated liver-targeted nanoplexes composing of poly-amidoamine (PAMAM) dendrimers, PEG, and lactobionic acid (PAMAM-PEG-Gal) 371 were applied to encapsulate and deliver siRNAs that specifically silence AEG-1 (PAMAM-AEG-1si) in the HFD-induced mouse model, resulting in a significant alleviation of liver damage and downregulated serum AST/ALT, liver weight, and TG/cholesterol levels. 370anscriptional co-activator with PDZ-binding motif Transcriptional co-activator with a PDZ-binding motif (TAZ), encoding a transcriptional co-activator sharing homology with Yesassociated protein (Yap), was found to bind to the PPXY motif through its WW domain. 372TAZ is considered to be related to mesenchymal differentiation and development of multiple organs. 373ang et al. observed elevated TAZ in the livers from NASH patients and MCD-induced murine models. 374In addition, AAV8-mediated liver-specific TAZ silencing was shown to reduce hepatic inflammation, hepatocyte death, and fibrosis in a NASH mouse model through repression of Indian hedgehog (Ihh)-mediated fibrogenic gene activation in HSCs. 374In the follow-up study, the same research group utilized TAZ siRNA conjugated with GalNAc (GalNAc-siTAZ) for therapeutic study.The results showed that GalNAc-siTAZ was able to prevent or even reverse NASH progression. 375gh-mobility group box 1 7][378] Plasma Hmgb1 level was found to be elevated in a diet-induced NASH mouse model 379 and positively correlated with the severity of liver fibrosis in NASH patients. 380Salvianolic acid B (SalB), a compound that inhibits Hmgb1 nuclear translocation and release, was demonstrated to protect against NAFLD in rats. 381hou et al. developed a stable mannose-modified LNP delivery system carrying HMGB1-siRNA (mLNP-siHMGB1) to achieve specific HMGB1 silencing in KCs via mannose receptors on their surfaces. 289,382The results showed that mLNP-siHMGB1 reduced Hmgb1 protein in the liver, shifted KCs to M2 phenotype, attenuated fibrosis, and restored liver function in the NASH mouse model. 289

OLX702A (by OliX Pharmaceuticals)
OLX702A is a GalNAc-conjugated asymmetric siRNA (asiRNA) drug with fewer off-target and side effects than other siRNAs. 383It was innovated by OliX Pharmaceuticals to target NASH-related genes found in a human NASH genome-wide association study (GWAS). 384The administration of OLX702A was shown to significantly reduce liver fat content in a non-human primate NASH model. 385ng non-coding RNAs lncRNA HULC (highly upregulated in liver cancer) expression was found to be upregulated in HFD-induced rat models. 386Shen et al.
demonstrated that siRNA plasmid targeting HULC in vivo significantly reduced lipid deposition, fibrosis, and hepatocyte apoptosis in the NAFLD rat model through the p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways. 386[389] miRNA mimic or inhibitor MicroRNA-132 miR-132 levels were found to be significantly increased in NAFLD patients and murine NASH models, while transgenic mice with overexpressed miR-132 exhibited liver steatosis and hyperlipidemia. 390iR-132 was first demonstrated to inhibit Sirt1 expression through directly binding to SIRT1 3ʹ-untranslated region (UTR) in adipocytes. 391Sirt1 has been reported to regulate various transcription factors involved in inflammation, lipid metabolism, and insulin secretion (e.g., p53, NF-kB, PPAR-a, PPAR-g, PPAR-g co-activator 1a [Pgc-1a], and liver X receptor [Lxr]) via its deacetylase activity in specific sites. 392,393Therefore, anti-miR-132-mediated SIRT1 derepression could be applied as a potential approach in intervening metabolismrelated diseases including NAFLD.Studies have shown that dietinduced obese (DIO) mice treated with anti-miR-132 displayed resolved liver steatosis as well as reduced liver FFA and serum LDL/ VLDL. 390The above data collectively suggest that the downregulation of miR-132 has the potential to impede NASH progression.Regulus Therapeutics tested oligonucleotide-based miR-132 antagonists in DIO, choline-deficient high-fat diet (CDHFD), and amylin liver NASH (AMLN) models, showing promising efficacies in treatments. 394croRNA-103/107 Differing by only one nucleotide, 395 miR-103 and miR-107 paralogs exist within the intron region of PANK, which encodes the pantothenate kinase (Pank). 396They were shown to be co-transcribed with this gene to regulate several target mRNAs involved in lipid and pyruvate metabolic pathways. 396Studies have shown that the levels of these two miRNAs were significantly upregulated in the livers of obese mice with steatosis, 397 which induce impaired glucose homeostasis and insulin sensitivity by inhibiting the expression of cavevolin-1, 398 a factor known to enhance insulin receptor signaling. 399,400Therefore, anti-miR-103/107 could function as an insulin sensitizer.A study conducted by Regulus Therapeutics showed that the administration of anti-miR-103/107 reduced TG level and liver steatosis in mice. 401Clinical trials of RG-125 (AZD4076), a GalNAc-conjugated anti-miR-103/107 designed for treating NAFLD/NASH, have been launched. 401In particular, the phase 1/2a study in T2D patients with NAFLD has been completed (NCT02826525), and a phase 1 study in patients with NASH is now under way (NCT02612662).
MicroRNA-22 miR-22 was previously reported as a tumor suppressor to regulate colon and liver cancer. 402Elevated miR-22 was observed in the serum of NAFLD patients. 403Further studies demonstrated that miR-22 expression was negatively correlated with Fgf21 levels in human or mice with fatty liver, as miR-22 directly targets FGFR1 3 0 UTR and downregulates FGF21 transcription through decreasing the recruitment of PPAR-a and PGC-1a. 404In addition, miR-22 was also reported to affect lipogenesis and production of pro-inflammatory cytokines through silencing SIRT1 transcription, 405 suggesting that miR-22 inhibition may have therapeutic potential for harnessing NAFLD and obesity by manipulating the metabolic gene-expression landscape.An anti-miR-22 drug candidate, APT-110, was shown to increase insulin sensitivity and effectively reduce hepatic steatosis in mice, suggesting the potential application in NAFLD treatment. 406esalis Therapeutics is currently leading a pre-clinical study to inhibit miR-22 using LNA-based anti-miR-22 (RES-010) for treating NASH/ NAFLD. 407,408croRNA-33 miR-33a was identified as an intronic miRNA located within SREBP2, encoding sterol regulatory element binding factor 2 (Srebf2), a transcriptional regulator targeting the expression of cellular cholesterol transporters in cholesterol metabolism. 409Studies have shown that the regulation of glucose homeostasis was improved and the development of fibrosis and inflammation was slowed in a hepatic miR-33a deficiency conditional knockout mouse model. 410In addition to miR-33a in mice, miR-33b is located in the intron of SREBP1 in humans, 411 which is a crucial regulator in hepatic FA synthesis. 412ecently, studies have shown that anti-miR-33 treatments, especially anti-miR-33b, ameliorated liver dysfunction and improved the serum and liver lipid profile in Gubra amylin NASH (GAN) diet-induced mice with miR-33b knockin in the intron of SREBP1. 413€ uppel-like factor 11 Serum miR-10b levels in NASH patients have been shown to negatively correlate with the lobular inflammation score. 414Its expression was also observed to be significantly lower in the livers of mice with HFD-induced IR. 415 Mechanistically, miR-10b-5p was shown to upregulate RTK (encoding receptor tyrosine kinase) expression through suppressing key transcription factor Krüppel-like factor 11 (Klf11) in interstitial cells of Cajal (ICCs) or pancreatic b cells. 416,417In pre-clinical studies, injection of the miR-10b-5p mimic successfully improved glucose homeostasis and gastrointestinal (GI) motility in mice, 417 indicating the therapeutic potential of miR-10b-5p mimic in treating metabolic diseases.Led by RosVivo Therapeutics, the development of the miR-10b-5p mimic (RSVI-301) is currently under way for a group of metabolic diseases including NAFLD, T2D, obesity, and GI motility. 418

CONCLUSIONS AND FUTURE DIRECTIONS
Thanks to considerable advances in demonstrating underlying links between NASH and various pathological processes including dysregulated lipid metabolism, IR, inflammation, and fibrosis, abundant potential therapeutic targets have been uncovered.However, based on the results from particular clinical trials, Glp-1 analogs were found only to prevent the progression of liver steatosis but not to resolve the pathological changes especially in middle-to-late stage.Relying on advanced chemical modification strategies and a specific liver-targeted delivery system, oligonucleotide drugs are now becoming safe, stable, and selective in therapeutic applications of liver metabolic diseases, including NASH.However, unlike some of the metabolic diseases which are driven by single gene abnormality, NAFLD/ NASH is considered a complex syndrome caused by alterations in multiple parameters.Therefore, one key question in this field is whether satisfactory therapeutic efficacy against NASH could be achieved by blocking any single target-in other words, whether multi-target therapies could be applied by combinatory administration of oligonucleotide drugs with conventional drugs.Besides, in addition to the involvement of hepatocytes, other cell types including HSCs and KCs also participate in these processes.However, effective oligonucleotide drug delivery for hepatic non-parenchymal cells remains an obstacle.Therefore, continuous inquiries into more accurate and selective delivery systems are needed.Depending on the results of current clinical and pre-clinical studies on oligonucleotide drugs, the effectiveness and safety of this emerging therapeutic strategy are believed to be open to further improvement, ultimately benefiting NASH patients.

Table 2 .
Oligonucleotide therapeutics for NASH in clinical trials 357,358Sato et al. showed successful HSC-specific delivery of siRNA targeting rat HSP47 homolog through vitamin A-coupled liposomes, which alleviated liver fibrosis and resolved collagen deposition in multiple liver disease models induced by dimethylnitrosamine (DMN), CCl 4 , and bile duct ligation,