Despite wide recognition as a disease of pandemic proportions, effective treatments for nonalcoholic fatty liver disease (NAFLD) remain elusive. Most of the current clinical programs aim to reduce hepatic fat accumulation and, thus, prevent downstream inflammation and fibrosis. To date, this therapeutic approach has helped identify a potential disconnect between steatosis reduction and disease resolution. Mounting preclinical evidence indicates liver inflammation may play a major role in steatosis development and fibrosis but has not garnered the same clinical representation. This may be owing to deficiencies in standard therapeutic modalities that limit their application in NAFLD. RNA interference (RNAi) is an attractive approach to targeting liver inflammation owing to its clinical safety profile, target specificity, and limited biodistribution. In this study, we characterize a simple cholesterol–short-interfering RNA (siRNA) conjugate system targeting Tnf mRNA in liver macrophages for the treatment of NAFLD. First, we observed delivery and anti-inflammatory activity in an acute liver inflammation model. In a follow-up murine NAFLD model, we observed total prevention of nearly all hallmarks of this disease: steatosis, inflammation, and fibrosis. This simple conjugate siRNA delivery system may be the first to show RNAi activity in liver macrophages and provide evidence for a novel therapeutic approach to inflammation in NAFLD.
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References
1.
FriedmanSL, Neuschwander-TetriBA, RinellaM and SanyalAJ. (2018). Mechanisms of NAFLD development and therapeutic strategies. Nat Med, 24:908–922.
2.
LucasC, LucasG, LucasN, Krzowska-FirychJ and TomasiewiczK. (2018). A systematic review of the present and future of non-alcoholic fatty liver disease. Clin Exp Hepatol, 4:165–174.
3.
PerumpailBJ, KhanMA, YooER, CholankerilG, KimD and AhmedA. (2017). Clinical epidemiology and disease burden of nonalcoholic fatty liver disease. World J Gastroenterol, 23:8263–8276.
4.
Albhaisi SAM and SanyalAJ. (2021). New drugs for NASH. Liver Int 41 Suppl, 1:112–118.
5.
YounossiZM, LoombaR, RinellaME, BugianesiE, MarchesiniG, Neuschwander-TetriBA, SerfatyL, NegroF, CaldwellSH, et al. (2018). Current and future therapeutic regimens for nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Hepatology, 68:361–371.
6.
FriedmanSL, RatziuV, HarrisonSA, AbdelmalekMF, AithalGP, CaballeriaJ, FrancqueS, FarrellG, KowdleyKV, et al. (2018). A randomized, placebo-controlled trial of cenicriviroc for treatment of nonalcoholic steatohepatitis with fibrosis. Hepatology, 67:1754–1767.
7.
RatziuV, SanyalA, HarrisonSA, WongVW, FrancqueS, GoodmanZ, AithalGP, KowdleyKV, SeyedkazemiS, et al. (2020). Cenicriviroc treatment for adults with nonalcoholic steatohepatitis and fibrosis: final analysis of the phase 2b CENTAUR study. Hepatology, 72:892–905.
8.
KazankovK, JorgensenSMD, ThomsenKL, MollerHJ, VilstrupH, GeorgeJ, SchuppanD and GronbaekH. (2019). The role of macrophages in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Nat Rev Gastroenterol Hepatol, 16:145–159.
9.
KrenkelO, PuengelT, GovaereO, AbdallahAT, MossanenJC, KohlheppM, LiepeltA, LefebvreE, LueddeT, et al. (2018). Therapeutic inhibition of inflammatory monocyte recruitment reduces steatohepatitis and liver fibrosis. Hepatology, 67:1270–1283.
10.
MiuraK, YangL, van RooijenN, OhnishiH and SekiE. (2012). Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2. Am J Physiol Gastrointest Liver Physiol, 302:G1310–1321.
11.
HuangW, MetlakuntaA, DedousisN, ZhangP, SipulaI, DubeJJ, ScottDK and O'DohertyRM. (2010). Depletion of liver Kupffer cells prevents the development of diet-induced hepatic steatosis and insulin resistance. Diabetes, 59:347–357.
12.
RiveraCA, AdegboyegaP, van RooijenN, TagalicudA, AllmanM and WallaceM. (2007). Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis. J Hepatol, 47:571–579.
13.
Tosello-TrampontAC, LandesSG, NguyenV, NovobrantsevaTI and HahnYS. (2012). Kuppfer cells trigger nonalcoholic steatohepatitis development in diet-induced mouse model through tumor necrosis factor-alpha production. J Biol Chem, 287:40161–40172.
14.
TomitaK, TamiyaG, AndoS, OhsumiK, ChiyoT, MizutaniA, KitamuraN, TodaK, KanekoT, et al. (2006). Tumour necrosis factor alpha signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice. Gut, 55:415–424.
15.
AlAsfoorS, RohmTV, BoschAJT, DervosT, CalabreseD, MatterMS, WeberA and Cavelti-WederC. (2018). Imatinib reduces non-alcoholic fatty liver disease in obese mice by targeting inflammatory and lipogenic pathways in macrophages and liver. Sci Rep, 8:15331.
16.
WandrerF, LiebigS, MarhenkeS, VogelA, JohnK, MannsMP, TeufelA, ItzelT, LongerichT, et al. (2020). TNF-Receptor-1 inhibition reduces liver steatosis, hepatocellular injury and fibrosis in NAFLD mice. Cell Death Dis, 11:212.
17.
FeaginsLA, FloresA, ArriensC, ParkC, CrookT, ReimoldA and BrownG. (2015). Nonalcoholic fatty liver disease: a potential consequence of tumor necrosis factor-inhibitor therapy. Eur J Gastroenterol Hepatol, 27:1154–1160.
18.
LikhitsupA, DundulisJ, AnsariS, PatibandlaS, HuttonC, KennedyK, HelzbergJH and ChhabraR. (2019). High prevalence of non-alcoholic fatty liver disease in patients with inflammatory bowel disease receiving anti-tumor necrosis factor therapy. Ann Gastroenterol, 32:463–468.
19.
TangKT, DufourJF, ChenPH, HernaezR and Hutfless.S (2020). Antitumour necrosis factor-alpha agents and development of new-onset cirrhosis or non-alcoholic fatty liver disease: a retrospective cohort. BMJ Open Gastroenterol, 7:e000349.
20.
CraigK, AbramsM and AmijiM. (2018). Recent preclinical and clinical advances in oligonucleotide conjugates. Expert Opin Drug Deliv, 15:629–640.
21.
SchluepT, LickliterJ, HamiltonJ, LewisDL, LaiCL, LauJY, LocarniniSA, GishRG and GivenBD. (2017). Safety, tolerability, and pharmacokinetics of ARC-520 injection, an RNA interference-based therapeutic for the treatment of chronic Hepatitis B virus infection, in healthy volunteers. Clin Pharmacol Drug Dev, 6:350–362.
22.
HouX, ZaksT, LangerR and DongY. (2021). Lipid nanoparticles for mRNA delivery. Nat Rev Mater, 6:1078–1094.
23.
BijsterboschMK, ManoharanM, DorlandR, WaarloIH, BiessenEA and van BerkelTJ. (2001). Delivery of cholesteryl-conjugated phosphorothioate oligodeoxynucleotides to Kupffer cells by lactosylated low-density lipoprotein. Biochem Pharmacol, 62:627–633.
24.
BijsterboschMK, RumpET, De VruehRL, DorlandR, van VeghelR, TivelKL, BiessenEA, van BerkelTJ and ManoharanM. (2000). Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation. Nucleic Acids Res, 28:2717–2725.
25.
WolfrumC, ShiS, JayaprakashKN, JayaramanM, WangG, PandeyRK, RajeevKG, NakayamaT, CharriseK, et al. (2007). Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat Biotechnol, 25:1149–1157.
26.
BrownCR, GuptaS, QinJ, RacieT, HeG, LentiniS, MaloneR, YuM, MatsudaS, et al. (2020). Investigating the pharmacodynamic durability of GalNAc-siRNA conjugates. Nucleic Acids Res, 48:11827–11844.
27.
BiscansA, ColesA, HarasztiR, EcheverriaD, HasslerM, OsbornM and KhvorovaA. (2019). Diverse lipid conjugates for functional extra-hepatic siRNA delivery in vivo. Nucleic Acids Res, 47:1082–1096.
28.
KosovrastiVY, NechevLV and AmijiMM. (2016). Peritoneal macrophage-specific TNF-alpha gene silencing in LPS-induced acute inflammation model using CD44 targeting hyaluronic acid nanoparticles. Mol Pharm, 13:3404–3416.
29.
YadavS, GandhamSK, PanicucciR and AmijiMM. (2016). Intranasal brain delivery of cationic nanoemulsion-encapsulated TNFalpha siRNA in prevention of experimental neuroinflammation. Nanomedicine, 12:987–1002.
30.
FosterDJ, BrownCR, ShaikhS, TrappC, SchlegelMK, QianK, SehgalA, RajeevKG, JadhavV, et al. (2018). Advanced siRNA designs further improve in vivo performance of GalNAc-siRNA conjugates. Mol Ther, 26:708–717.
31.
NairJK, AttarwalaH, SehgalA, WangQ, AluriK, ZhangX, GaoM, LiuJ, IndrakantiR, et al. (2017). Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates. Nucleic Acids Res, 45:10969–10977.
32.
FitzgeraldK, WhiteS, BorodovskyA, BettencourtBR, StrahsA, ClausenV, WijngaardP, HortonJD, TaubelJ, et al. (2017). A highly durable RNAi therapeutic inhibitor of PCSK9. N Engl J Med, 376:41–51.
33.
JanasMM, JiangY, SchlegelMK, WaldronS, KuchimanchiS and BarrosSA. (2017). Impact of oligonucleotide structure, chemistry, and delivery method on in vitro cytotoxicity. Nucleic Acid Ther, 27:11–22.
34.
JanasMM, JiangY, DuncanRG, HayesAN, LiuJ, KasperkovitzPV, PlackeME and BarrosSA. (2016). Exposure to siRNA-GalNAc conjugates in systems of the standard test battery for genotoxicity. Nucleic Acid Ther, 26:363–371.
35.
OsbornMF and KhvorovaA. (2018). Improving siRNA delivery in vivo through lipid conjugation. Nucleic Acid Ther, 28:128–136.
36.
MatsumotoM, HadaN, SakamakiY, UnoA, ShigaT, TanakaC, ItoT, KatsumeA and SudohM. (2013). An improved mouse model that rapidly develops fibrosis in non-alcoholic steatohepatitis. Int J Exp Pathol, 94:93–103.
37.
TasnimF, XingJ, HuangX, MoS, WeiX, TanMH and YuH. (2019). Generation of mature kupffer cells from human induced pluripotent stem cells. Biomaterials, 192:377–391.
38.
IredaleJP, ThompsonA and HendersonNC. (2013). Extracellular matrix degradation in liver fibrosis: biochemistry and regulation. Biochim Biophys Acta, 1832:876–883.
39.
TabriziM, BornsteinGG and SuriaH. (2010). Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease. AAPS J, 12:33–43.
40.
JamesBH, PapakyriacouP, GardenerMJ, GliddonL, WestonCJ and Lalor.PF (2021). The contribution of liver sinusoidal endothelial cells to clearance of therapeutic antibody. Front Physiol, 12:753833.
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