HEPATOLOGY ELSEWHERE. Antagonizing MicroRNA-122 and Treatment of Hepatitis C Virus Infection. Abstract. Comment EDITORS

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1 HEPATOLOGY ELSEWHERE EDITORS Kris Kowdley, Seattle, WA Geoffrey McCaughan, Newtown, Australia Christian Trautwein, Aachen, Germany Antagonizing MicroRNA-122 and Treatment of Hepatitis C Virus Infection Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME, et al. Therapeutic silencing of microrna-122 in primates with chronic hepatitis C virus infection. Science 2010;327: (Reproduced with permission.) Abstract The liver-expressed microrna-122 (mir-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. We found that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA)-modified oligonucleotide (SPC3649) complementary to mir-122 leads to long-lasting suppression of HCV viremia, with no evidence ofviralresistanceorsideeffectsinthetreatedanimals.furthermore, transcriptome and histological analyses of liver biopsies demonstrated derepression of target mrnas with mir-122 seed sites, down-regulation of interferon-regulated genes, and improvement of HCV-induced liver pathology. The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance. Comment Hepatitis C virus (HCV) infection is a major cause of liver cirrhosis and hepatocellular carcinoma worldwide. Current antiviral treatment consisting of pegylated interferon-alpha (IFN-a) and ribavirin is limited by resistance, adverse effects, and high costs. 1 Although the clinical development of novel antiviral compounds that target HCV protein processing has been shown to markedly improve sustained virological response, toxicity of the individual compounds and development of viral resistance remain major challenges. 2 Thus, novel antiviral strategies are urgently needed. Micro-RNAs (mirnas) are key regulators of gene expression at a posttranscriptional level. 3 Their biogenesis is now well characterized and involves the processing of a large primary transcript into a stemloop pre-mirna, ultimately leading to the mature single-stranded 22-nucleotide mirna. This functional mirna is assembled into an RNA-induced silencing complex (RISC) that invariably contains a member of the Argonaute protein family (Fig. 1). Once loaded, the active RISC can be directed toward its messenger RNA target to regulate, predominantly in a negative manner, its translation. 4 Besides targeting cellular messenger RNAs, mirnas were recently shown to interact with transcripts of viral origin. 5 The first description of such interactions revealed that mirnas of cellular origin could negatively regulate viral messenger RNAs. Furthermore, mammalian viruses have been shown to usurp the cellular mirna repertoire. One remarkable example of such usurpation is provided by HCV, which recruits the liver-specific mir-122 to enhance its replication. 6 In vivo, the impact of mirna for pathogenesis of HCV infection is more complex: analyzing liver biopsies from subjects with chronic hepatitis C who are undergoing IFN therapy, Sarasin-Filipowicz et al. showed no correlation of mir-122 expression with viral load but markedly decreased pretreatment mir-122 levels in subjects who had no virological response during later IFN therapy. 7 To truly assess the importance of mirnas as a therapeutic target requires the use of chemically modified antisense oligonucleotides complementary to the mirna to prevent its interaction with the target RNA. This approach was first established in vitro, 8 before it was shown that it was also very effective in preventing mirna action in mouse models. 9 The later study was carried out using mir-122 as a model, and it enabled the identification of several cellular targets, most of which are involved in the cholesterol biogenesis pathway, e.g., 3-hydroxy-3-methylglutaryl-coenzymeA reductase. Primarily, two types of chemistries have been used to modify the antisense oligonucleotide and increase its stability and/or uptake by cells: 2 0 -O-methylated oligonucleotides, usually coupled to a cholesterol group 9 and, more recently, oligonucleotides containing locked nucleic acid (LNA) residues (Fig. 1). A LNA antisense oligonucleotide binding to the 5 0 part of mir-122 (SPC3649; Santaris Pharma, Hoersholm, Denmark) has been shown to be efficient via mouse models in confirming that blocking mir-122 results in a decrease in cellular targets involved in cholesterol biogenesis. 10 Next, the inhibition of mir-122 and its effect on cholesterol levels was confirmed in nonhuman primates. 11 The stage was set for testing the real effect of blocking mir-122 on HCV infection in a primate model. Following up on these investigations, mirna-122 has now been shown to be a target for antiviral intervention. In a report this month in Science, Robert 1461

2 1462 HEPATOLOGY ELSEWHERE HEPATOLOGY, April 2010 Fig. 1. Model of the mode of action of the LNA oligonucleotide SPC3649. The oligonucleotide binds to the 5 0 part of the mature mir-122 with strong affinity due to the incorporation of some modified bases containing a ribose locked by an extra covalent liaison (shown above the LNA). The binding of the LNA prevents the incorporation of mir-122 into its effector RISC complex, which it normally uses to regulate both cellular genes and HCV RNA. Therefore, when the LNA is present, cellular targets of mir-122 have increased expression (which results in a decrease of cholesterol levels), whereas HCV replication goes down. The two effects of the inhibition of mir- 122 are not interrelated. Lanford and colleagues showed that the inhibition of mir-122 in chimpanzees leads to long-lasting suppression of HCV viremia (Fig. 1). 12 Using high and low doses of the SPC3649 oligonucleotide, they demonstrated that treatment of chronically HCV-infected animals with the LNA oligonucleotide results in a marked and sustained decrease of HCV RNA in both serum and liver. The sequestration of mir-122 by the LNA oligonucleotide was confirmed, as well as the strong reduction of free mir-122 levels for the highdose animals. No rebound in viremia was observed during the treatment, and no adaptive mutations were found in the mir-122 binding sites. The analysis of the liver transcriptome revealed a marked down-regulation of IFN-regulated genes, which confirmed that the endogenous IFN pathway in the liver was normalized after inhibition of HCV RNA. Finally, as expected, the antagonism of mir-122 resulted in a strong decrease in serum cholesterol (Fig. 1). Besides that effect, no measurable toxic effect in the liver could be attributed to SPC3649. What are the clinical implications of this landmark study? The results of Lanford et al. clearly show that antagonism of mir-122 by the LNA oligonucleotide SPC3649 leads to marked suppression of viremia in chronically HCV-infected chimpanzees and improvement of HCV-induced liver pathology. The prolonged virological response to SPC3649 treatment without HCV rebound suggests that targeting mirna-122 by antagonists holds promise as a novel antiviral therapy. A potential advantage compared to therapeutic strategies that target viral factors may be the high barrier to resistance, as demonstrated by the lack of rebound in viremia during the treatment and the lack of adaptive mutations in the two mir-122 seed sites of the HCV 5 0 noncoding region. Furthermore, conservation of both mir-122 seed sites in all HCV genotypes and subtypes suggests that such therapy will most likely be genotype-independent. A potential limitation of the therapeutic approach for the HCV-infected patient may be the delivery of the oligonucleotide, via frequent intravenous injections. Addressing this point in their discussion, the authors suggest that the safety profile and high stability of the LNA oligonucleotide in vivo combined with the prolonged suppression of viremia beyond treatment could result in less-frequent dosing after viral suppression is attained. Safety is another potential concern for an approach that targets a host factor involved in regulation of several genes. Although an extensive analysis of clinical symptoms, clinical chemistry, and histopathology in this small cohort of chimpanzees did not reveal any evidence of serious adverse effects, more-detailed investigations in humans are required to rule out potential adverse effects. Taken together, this study demonstrates the feasibility and safety of prolonged administration of an LNA oligonucleotide drug that antagonizes the function of a specific mirna in a clinically relevant infectious disease model. Clinical trials are the next step to demonstrate the efficacy and safety of this approach in the HCV-infected patient. SÉBASTIEN PFEFFER Architecture et Réactivité de l Acide Ribonucléique Université de Strasbourg Institut de Biologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique Strasbourg, France THOMAS F. BAUMERT Institut National de la Santé et de la Recherche Médicale U748, Université de Strasbourg, Strasbourg, France Pôle Hépato-digestif, Nouvel Hôpital Civil Strasbourg, France

3 HEPATOLOGY, Vol. 51, No. 4, 2010 HEPATOLOGY ELSEWHERE 1463 References 1. Tai AW, Chung RT. Treatment failure in hepatitis C: mechanisms of non-response. J Hepatol 2009;50: Hezode C, Forestier N, Dusheiko G, Ferenci P, Pol S, Goeser T, et al. Telaprevir and peginterferon with or without ribavirin for chronic HCV infection. N Engl J Med 2009;360: Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116: Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by mirnas: how many mechanisms? Trends Cell Biol 2007;17: Gottwein E, Cullen BR. Viral and cellular micrornas as determinants of viral pathogenesis and immunity. Cell Host Microbe 2008;3: Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 2005;309: Sarasin-Filipowicz M, Krol J, Markiewicz I, Heim MH, Filipowicz W. Decreased levels of microrna mir-122 in individuals with hepatitis C responding poorly to interferon therapy. Nat Med 2009;15: Meister G, Landthaler M, Dorsett Y, Tuschl T. Sequence-specific inhibition of microrna- and sirna-induced RNA silencing. RNA 2004; 10: Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, et al. Silencing of micrornas in vivo with antagomirs. Nature 2005;438: Elmen J, Lindow M, Silahtaroglu A, Bak M, Christensen M, Lind-Thomsen A, et al. Antagonism of microrna-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mrnas in the liver. Nucleic Acids Res 2008;36: Elmen J, Lindow M, Schutz S, Lawrence M, Petri A, Obad S, et al. LNA-mediated microrna silencing in non-human primates. Nature 2008;452: Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME, et al. Therapeutic silencing of microrna-122 in primates with chronic hepatitis C virus infection. Science 2010;327: Copyright VC 2010 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Financial support: This work was supported by Centre National de la Recherche Scientifique and Institut National de la Santé et de la Recherche Médicale, France; the European Union (ERC-2008-AdG HEPCENT, EU Interreg-IV-2009 HEPATO-REGIO-NET); Agence Nationale de la Recherche (ANR-05-CEXC-008), ANRS France (2008/354), Paris; and the Else-Kröner- Fresenius Stiftung, Bad Homburg (P17/07//A83/06). Potential conflict of interest: Nothing to report. Patatin-Like Phospholipase Domain Containing 3: A Case in Point Linking Genetic Susceptibility for Alcoholic and Nonalcoholic Liver Disease Tian C, Stokowski RP, Kershenobich D, Ballinger DG, Hind DA. Variant in PNPLA3 is associated with alcoholic liver disease. Nat Genet 2009;42: Available at (Reprinted with permission.) Abstract Two genome-wide association studies (GWAS) have described associations of variants in PNPLA3 with nonalcoholic fatty liver and plasma liver enzyme levels. We investigated the contributions of these variants to liver disease in Mestizo subjects with a history of alcohol dependence. We found that rs in PNPLA3 is strongly associated with alcoholic liver disease and clinically evident alcoholic cirrhosis (unadjusted OR ¼ 2.25, P ¼ ; ancestry-adjusted OR ¼ 1.79, P ¼ ). Comment Alcoholic liver disease (ALD) includes a spectrum of histopathological injury ranging from simple fatty liver or steatosis through alcoholic hepatitis, hepatic fibrosis and cirrhosis, to end-stage liver disease. Fatty liver develops in more than 90% of drinkers whereas only 8%-15% of heavy drinkers (>40 g/day) develop cirrhosis. A strong association exists between the amount of alcohol intake and consumption patterns and ALD. In addition, factors such as sex, genetic background, and environmental factors (e.g., nutrition) appear to play an important role in determining which heavy drinker develops cirrhosis. Evidence supporting a role for genetic factors comes from twin studies 1 and from the observation that the death rate from ALD is subject to wide interethnic variation that is not entirely explained by variations in the prevalence of alcohol abuse. 2,3 For example, Hispanics appear to be at particularly high risk. In the United States, the death rate (per 100,000) from ALD among men was reported to be the highest in Hispanic whites (12.6) followed by non-hispanic African Americans (7.4), non-hispanic whites (5.2), and Hispanic African Americans (1.8). 4 Patients with all stages of ALD often have coexistent risk factors for nonalcoholic fatty liver disease (NAFLD) including obesity and hyperglycemia. 5 NAFLD also follows similar histopathological sequelae to ALD from fatty liver, through inflammation to fibrosis and cirrhosis, and appears to share many pathogenic mechanisms with ALD including oxidative and endoplasmic reticulum mediated stress and endotoxinmediated cytokine release. 6 Furthermore, NAFLD shows similar interethnic variation to ALD, with Hispanics showing the highest prevalence of disease and African Americans the lowest. 7 Accordingly, it seems likely that genetic factors predisposing an individual to NAFLD may also play a role in determining the risk of ALD. This article from Tian and colleagues provides the first definitive evidence that this is indeed the case, by showing an association between ALD and a variant allele in the gene encoding PNPLA3 (patatin-like phospholipase domain containing 3) recently associated with NAFLD and/or raised serum aminotransferases in two recent genome-wide association studies (GWAS). 8,9 Romeo et al. demonstrated that in NAFLD, the PNPLA3 allele rs (C!G) showed a strong association with increased hepatic fat, as determined

4 1464 HEPATOLOGY ELSEWHERE HEPATOLOGY, April 2010 Genotype Table 1. Association of PNPLA3 Allele rs738409[g] with Alcoholic Cirrhosis Heavy Drinkers with Normal Liver Blood Tests (n 5 182) Alcoholic Cirrhosis (n 5 266) Odds Ratio CC 119 (65%) 173 (47.2%) 1 CG 60 (33%) 170 (46.5%) 1.95 ( ) GG 3 (1.6%) 23 (6.3%) 7.34 ( ) Chi-squared 2 for trend P < OR ¼ 2.2 (95% CI ¼ ; P ¼ ) for possession of G allele. OR ¼ 5.57 (95% CI ¼ ; P ¼ ) for GG genotype. by proton magnetic resonance spectroscopy ( 1 H-MRS) (P ¼ ), and plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in Hispanics. 9 The G allele, which is more prevalent in Hispanics (49%) than in African Americans (17%) and European Americans (23%) with NAFLD, was associated with a two-fold higher hepatic fat content in individuals homozygous for the G allele compared to CC homozygotes. In African Americans, possession of another PNPLA3 allele (rs [T]) was associated with an 18% lower hepatic fat content compared to GG homozygotes (P ¼ ), and this allele was extremely rare in European Americans (0.3%) and Hispanics (0.8%) compared to African Americans (10.4%). The rs variant has also been associated with 1 H-MRS determined liver fat content in a Finnish study, 8 and ALT and AST levels in an independent GWAS 10 and in another case control study in European White and Indian Asian populations. 11 PNPLA3 encodes for adiponutrin, a transmembrane protein highly expressed in the liver and adipose tissues. Adiponutrin is primarily found in the brown and white adipose tissues in animals. 12 In humans, its expression in subcutaneous and intra-abdominal adipose tissue strongly correlates with obesity, 13 whereas hepatic messenger RNA expression correlates with obesity and liver fat content. 8 The biochemical function of adiponutrin is uncertain, but is considered to have lipogenic transacetylase activity likely facilitating energy mobilization and lipid storage in adipose tissue and liver. 13,14 The variant rs238409[g] substitution changes codon 148 from a highly conserved isoleucine residue in vertebrates to methionine. The mutation is located in a putative splicing silencer domain, and thus it may have a gene regulatory role. 15 The functional significance of this mutation and the uncertainty of how it might influence the loss or gain of protein function with relevance to liver disease are other important unknowns. It can be envisaged that this mutation may act as a gain of function, enhancing lipid accumulation in the liver and as a result may be an indicator of mild hepatocyte injury/inflammation due to its association with liver aminotransferases. 10 Hind s group set out to examine whether variations in the PNPLA3 gene associated with NAFLD also play a role in determining liver disease susceptibility in Mestizo (mixed European and native American ancestry) individuals from Mexico City with a history of heavy drinking. They studied three groups of drinkers: those with clinically normal liver function (control, n ¼ 305), those with abnormal liver function ( intermediate ALD, n ¼ 434), and those with clinically evident cirrhosis (n ¼ 482). Genotyping was performed for the two nonsynonymous variants rs and rs identified in the NAFLD GWAS study, 9 as well as 15 common tagging singlenucleotide polymorphisms (SNPs) from the PNPLA3 region, 291 SNPs for assessing global ancestry, 16 ancestry-informative markers flanking the PNPLA3 region for assessing local ancestry, and 7 SNPs previously reported to be associated with cirrhosis in patients with hepatitis C. Analysis used likelihood ratio tests from logistic regression adjusted for age, alcohol intake, and duration and their interactions, and controlled for global ancestry with principle component analysis estimation. For the rs SNP, comparing patients with cirrhosis with controls gave an unadjusted odds ratio of 2.25 per G allele (P ¼ ) and an ancestry-adjusted odds ratio of 1.79 per G allele (P ¼ ). All common haplotypes containing the G allele were more prevalent in individuals with cirrhosis compared to control drinkers. There was no difference in genotype distributions between intermediate patients with ALD and controls after adjusting for global and local ancestry. Further analysis suggested that the rs variant accounts for 49% of the observed ancestry-related difference in cirrhosis susceptibility. Moreover, the G allele also showed a trend toward an association with increasing disease severity (frequencies of 0.70, 0.75, and 0.77 in Child-Pugh class A, B, and C, respectively), suggesting that this allele may also play a role in determining prognosis and clinical outcome. The rare variant rs (t) reported by Romeo et al. 9 was detected in both cirrhotic and control groups, but the very low frequency (0.0002) meant the study had insufficient power to detect any association with disease. No association was observed with any SNPs previously reported to be associated with hepatitis C related cirrhosis. The authors conclude that their study, taken together with evidence from other studies, supports the view that rs is an independent risk factor for liver dysfunction in fatty liver diseases.

5 HEPATOLOGY, Vol. 51, No. 4, 2010 HEPATOLOGY ELSEWHERE 1465 Indeed, recent data from Day s group, using a realtime fluorescent allele-specific system (K-Biosciences, Essex, UK), replicate these findings in a UK cohort to provide further evidence of rs738409(g) association with ALD (Table 1; C.P. Day et al., unpublished data). From what is known of the function of adiponutrin, these data strongly suggest that altered lipid processing plays a key role in the pathogenesis of progressive liver disease and provide further support for common pathogenic pathways in ALD and NAFLD. Clearly understanding the function of adiponutrin and its role in the pathogenesis of advanced fatty liver diseases is now the focus of considerable attention because this may lead to therapeutic advances for these common liver diseases for which there are currently no effective treatments available. For now, PNPLA3 genotyping offers the potential to identify individuals at increased risk of developing ALD and NAFLD providing the opportunity for targeted interventions. DEVANSHI SETH 1 ANN K. DALY 2 PAUL S. HABER 3 CHRISTOPHER P. D AY 2 1 Centenary Institute and Drug Health Services Royal Prince Alfred Hospital, University of Sydney Camperdown, NSW, Australia 2 Institute of Cellular Medicine, Newcastle University Newcastle upon Tyne, UK 3 Drug Health Services, Royal Prince Alfred Hospital Sydney South West Area, Health Service; University of Sydney, Camperdown, NSW, Australia References 1. Reed T, Page WF, Viken RJ, Christian JC. Genetic predisposition to organ-specific endpoints of alcoholism. Alcohol Clin Exp Res 1996;20: Caetano R, Clark CL. Trends among alcohol related problems among whites, blacks, and Hispanics: Alcohol Clin Exp Res 1998; 22: Stinson FS, Grant BF, Dufour MC. The critical dimension of ethnicity in liver cirrhosis mortality statistics. Alcohol Clin Exp Res 2001;25: Said A, Williams J, Holden J, Remington P, Musat A, Lucey MR. The prevalence of alcohol-induced liver disease and hepatitis C and their interaction in a tertiary care setting. Clin Gastroenterol Hepatol 2004;2: Raynard B, Balian A, Fallick D, Capron F, Bedossa P, Chaput JC, et al. Risk factors of brosis in alcohol-induced liver disease. HEPATO- LOGY 2002;35: Day CP. From fat to inflammation. Gastroenterology 2006;130: BrowningJD,KumarKS,SaboorianMH,TheileDL.Ethnicdifferences in the prevalence of cryptogenic cirrhosis. Am J Gastroenterol 2004;99: Kotronen A, Johansson LE, Johansson IM, Roos C, Westerbacka J, Hamsten A, et al. A common variant in PNPLA3, which encodes adiponutrin, is associated with liver fat content in humans. Diabetologia 2009;52: Romeo S, Kozlitina J, Xing C, Pertsemlidis A, Cox D, Pennacchio LA, et al. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 2008;40: Kollerits B, Coassin S, Kiechl S, Hunt SC, Paulweber B, Willeit J, et al. A common variant in the adiponutrin gene influences liver enzyme levels. J Med Genet 2009; doi: /jmg Yuan X, Waterworth D, Perry JRB, Lim N, Song K, Chambers JC, et al. Population-based genome-wide association studies reveal six loci influencing plasma levels of liver enzymes. Am J Hum Genet 2008;83: Lake AC, Sun Y, Li JL. Expression, regulation, and triglyceride hydrolase activity of adiponutrin family members. J Lipid Res 2005;46: Baulande S, Lasnier F, Lucas M, Pairault J. Adiponutrin, a transmembrane protein corresponding to a novel dietary-and obesity-linked mrna specifically expressed in the adipose lineage. J Biol Chem 2001; 276: Wilson PA, Gardner SD, Lambie NM, Commans SA, Crowther DJ. Characterization of the human patatin-like phospholipase family. J Lipid Res 2006;47: Yuan HY, Chiou JJ, Tseng WH, Liu CH, Liu CK, Lin YJ, et al. FASTSNP: an always up-to-date and extendable service for SNP function analysis and prioritization. Nucleic Acids Res 2006;34:W635-W641. Copyright VC 2010 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Nothing to report.