HK1200876B - A single nucleotide polymorphism on chromosome 15 that predicts hcv treatment responses - Google Patents

Description

Single nucleotide polymorphism on chromosome 15 for predicting HCV therapeutic response

The present invention relates to methods for predicting the response of a patient infected with Hepatitis C Virus (HCV) to a drug therapy (pharmacological treatment).

Hepatitis C Virus (HCV) is responsible for most of the chronic liver diseases worldwide and causes 70% of chronic hepatitis cases in industrialized countries. The global prevalence of Chronic Hepatitis C (CHC) is estimated to be 3% on average (in the range 0.1% to 5%), and 1.7 million chronic carriers (270 million in the us and 500 million in western europe) [1,2] are estimated globally. Approximately 1/5 chronically infected patients with HCV eventually develop cirrhosis of the liver, which may lead to liver failure and hepatocellular carcinoma [2 ]. HCV-related liver failure is one of the leading causes of liver transplantation today.

The current standard of care (SOC) for the first treatment of patients infected with chronic hepatitis c is combination therapy with interferon alpha (PEG-IFN) plus Ribavirin (RBV) conjugated with polyethylene glycol. In patients chronically infected with HCV genotype1, which represents the majority of HCV-infected patients, the response to PEG-IFN remains suboptimal with a Sustained Virological Response (SVR) rate of between 42-52% [4, 5,6 ]. There is a real need for new treatment options for this patient population. Interferon alpha (IFN) was the first drug to show biological activity against HCV. The interferon alpha-2 a molecule has been chemically modified by covalently binding a branched methoxy polyethylene glycol moiety [9 ]. PEG-IFN has reduced systemic clearance and approximately 10-fold increased serum half-life compared to interferon alpha-2 a, and thus circulates much longer in the blood than the parent compound. Subsequent evaluations in three large clinical trials of more than 1400 patients showed that treatment with 180gPEG-IFN once weekly (qw) was more effective than treatment with IFN three times weekly [10 ].

Ribavirin is a guanosine analog that inhibits the replication of a wide range of RNA and DNA viruses in vitro [11 ]. The mechanism by which RBV acts as an antiviral substance has not been completely established, although this may involve alterations in the nuclear nucleotide pool and inhibition of viral RNA synthesis [12 ]. RBV monotherapy has little or no effect on HCV replication, but can result in normalization of serum alanine Aminotransferase (ALT) activity and improvement of liver histology. However, relapse occurs in almost all patients treated with RBV alone [13,14 ]. It has been found that combining RBV with PEG-IFN is more effective in treating CHC than PEG-IFN monotherapy. In a large clinical trial of 1121 patients for all genotypes, a sustained virological response was achieved in 53% of patients treated with PEG-IFN plus RBV compared to 29% treated with PEG-IFN alone (SVR) [10 ].

The likelihood of achieving a Sustained Virological Response (SVR) varies with the group of patients and viral factors. For example, younger patients, caucasian and Asian patients, and individuals who do not have advanced liver fibrosis, are more likely to clear infection with HCV after treatment [15-18 ]. Similarly, patients infected with HCV genotype 2 or 3, but not genotype1, and those with low baseline HCV rna levels in serum had the best chance of cure [4-6,16,18 ].

A more accurate prediction of SVR is now possible only after initiation of treatment. Regardless of the genotype of HCV, individuals who clear HCV rna after 4 or 12 weeks of treatment have a better chance of achieving SVR than those with persistent viremia [19 ]. The rapid virological response (RVR, no hcv rna detected at week 4) is a strong predictor of SVR; conversely, the inability to achieve early virological responses (EVR, decline of hcv rna by more than two log values at week 12) is a strong predictor of no response independent of pretreatment profile [20 ].

Being able to predictably distinguish between potential responders and non-responders to the standard of care will have a large impact on the care of patients with chronic hepatitis c. In addition to host and viral factors, the genetic diversity of the host also affects the response to standard of care treatments [21 ]. Recent evidence from genome-wide association studies suggests that Single Nucleotide Polymorphisms (SNPs) in the promoter region of the IL-28b gene have a strong influence on the probability of SVR in patients treated with PEG-IFN plus ribavirin [22-24 ].

It has recently been found that in patients infected with hepatitis c virus genotype 1(HCV-1) or genotype 4(HCV-4), if the HCV rna level of the patient becomes undetectable in as little as two weeks after treatment, a beneficial response to treatment comprising interferon alpha, ribavirin and HCV polymerase inhibitors (triple therapy) can be predicted. The correlation between showing a rapid virological response at the beginning of triple therapy treatment patients-2 weeks (RVR2) and achieving a Sustained Virological Response (SVR) at the end of triple therapy treatment patients is disclosed in commonly owned us patent application USSN61/138,585 filed on 18.12.2008, which is incorporated herein by reference in its entirety.

The present invention is based on the discovery that: an association between a single nucleotide polymorphism on human chromosome 15 and RVR2 in patients treated with an interferon-based regimen that includes a direct acting antiviral agent, such as an HCV protease inhibitor. In one embodiment, the present invention provides a method for predicting the response of a human subject infected with HCV to treatment with peginterferon alfa-2a, ribavirin and a direct acting antiviral agent, comprising providing a sample from the human subject and identifying the nucleotide present at the single nucleotide polymorphism rs12148487, wherein a subject having at least one a allele present at rs12148487 has a higher probability of achieving RVR2 for the treatment relative to a subject having two G alleles present at rs 12148487.

FIG. 1 study design of clinical trial NV 21075. PEG-IFN ═ pegylated interferon alpha-2 a; RBV ═ ribavirin; q8h ═ three times per day; q12h is twice daily.

Figure 2. percentage of patients with virological response by week 12.

FIG. 3. change in mean HCVRNA (log10IU/ml) from baseline by week 12.

Figure 4 percentage of CC patients and non-CC patients in the rs12979860SNP with virological response by week 12.

FIG. 5 Whole genome Association results of (A) RVR2 and (B) RVR2 after chromosome adjustment of rs12979860 in the NV21075 study population.

FIG. 6 is a bar graph showing the association between RVR2 and rs12148487 genotypes across 4 treatment groups in the NV21075 clinical trial.

To facilitate an understanding of the present invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms such as "a," "an," and "the" are not intended to refer to only a singular entity, but also include the general types of entities that may be used in the specific examples of an example. The terms used herein are used to describe specific embodiments of the invention, but their use is not intended to limit the invention unless otherwise summarized in the claims.

The term "response" to interferon therapy is the desired response to administration of an agent. Virological endpoints included the following: "Rapid virological response-2 weeks" (RVR2) defined as the absence of detectable HCVRNA in serum after 2 weeks of treatment (byAmpliPrep/HCVTestv1.0, detection limit 15 IU/mL); "Rapid virological response-4 weeks" (RVR4) defined as the failure to detect HCVRNA in serum after 4 weeks of treatment (byAmpliPrep/HCVTestv1.0, detection limit 15 IU/mL); an "early virological response" (EVR), defined as the 2 log unit drop in serum hcv rna from baseline by week 12 (byAmpliPrep/HCVTest, v1.0, limit of quantitation 43IU/mL), complete EVR (cEVR), defined as undetectable HCVRNA in serum after 12 weeks of treatment (byAmpliPrep/HCVTestv1.0, detection limit 15 IU/mL); and a "sustained virological response" (SVR) defined as the absence of detectable HCV RNA in the serum at the end of the 12-week untreated follow-up period (SVR-12) or the 24-week untreated follow-up period (SVR-24) ((SVR-12))<15 IU/mL). The term "extended rapid virological response" (ERVR) means that HCV RNA could not be detected during the treatment period of 4 to 20 weeks: (ERVR) ((R))<15IU/mL)。

The term "sample" or "biological sample" refers to a tissue or fluid sample isolated from an individual, including, but not limited to, for example, biopsies, plasma, serum, whole blood, spinal fluid, lymph fluid, external sections of the skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. The sample also includes a sample of in vitro cell culture components (including but not limited to conditioned medium taken from cell growth in culture medium, cells putatively infected with a virus, recombinant cells, and cell components).

The terms "interferon" and "interferon- α" are used interchangeably herein and refer to a highly homologous, species-specific family of proteins that inhibit viral replication and cellular proliferation, as well as modulate immune responsesInterferon a, recombinant interferon α -2a as obtained from huffman-larovi ltd, Nutley, n.jinterferon-A, recombinant interferon α -2c as obtained from Boehringer Ingelheim pharmaceutical, Inc, Ridgefield, Connα 2 Interferon, Interferon α -nl, which is a purified mixture of the native α Interferon, as obtained from Sumitomo, JapanOr from Glaxo-Wellcomeltd, London, UKInterferon α -nl (ins), or consensus α interferons such as those described in U.S. patent nos. 4,897,471 and 4,695,623 (especially those of examples 7,8, or 9) and specific interferons obtained from Amgen, incThe product, or interferon α -n3, which is a natural α -interferon mixture made by interferon sciences, and said interferon α -n3 is available from purdue frederickco, norwalk, conn.

The terms "pegylated interferon," "pegylated interferon α," and which are used interchangeably herein, refer to polyethylene glycol modified conjugates of interferon α, preferably of interferons α -2a and α -2b exemplary suitable pegylated interferons α include, but are not limited toAnd

the term "ribavirin" refers to the compound 1- ((2R,3R,4S,5R) -3, 4-dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl) -1H- [1,2,4]Triazole-3-carboxylic acid amides which are synthetic non-interferon-induced broad-spectrum antiviral nucleoside analogs and are known by the nameAndcan be obtained.

"direct acting antiviral agents" exert specific antiviral effects independent of immune function. Examples of direct acting antiviral agents for HCV include, but are not limited to, protease inhibitors, polymerase inhibitors, NS5A inhibitors, IRES inhibitors, and helicase inhibitors.

The currently recommended first-line treatment for patients with chronic hepatitis c is pegylated interferon alfa in combination with ribavirin for 48 weeks in patients carrying genotype1 or 4 virus and for 24 weeks in patients carrying genotype 2 or 3 virus. Treatment with ribavirin has been found to be more effective than interferon alpha monotherapy in patients who relapse after one or more courses of interferon alpha treatment, as well as in previously untreated patients. However, ribavirin displays significant side effects, which include teratogenicity and carcinogenicity. Furthermore, ribavirin causes hemolytic anemia that may be associated with accumulation of ribavirin triphosphate in erythrocytes, requiring reduced doses or discontinuation of ribavirin therapy in about 10% to 20% of patients. Therefore, to reduce the cost of treatment and the incidence of adverse events, it is desirable to adjust treatment to shorter durations without reducing efficacy. For genotype1 patients treated with pegylated interferon alfa and ribavirin, the shortened "treatment duration" may be, for example, 24 weeks. For genotype1 patients treated with pegylated interferon alfa and ribavirin in combination with direct acting antiviral agents, the shortened treatment duration can be as short as 8 weeks, 12 weeks, or 16 weeks.

The terms "danoprevir", "RG 7227", "RO 5190591" and "ITMN-191" are used interchangeably and refer to the macrocyclic peptidomimetic inhibitor of hcv ns3/4A protease (macropeptide mimetic inhibitor), 4-fluoro-1, 3-dihydro-isoindole-2-carboxylic acid (Z) - (1S,4R,6S,14S,18R) -14-tert-butoxycarbonylamino-4-cyclopropanesulfonylaminocarbonyl-2, 15-dioxo-3, 16-diaza-tricyclo [14.3.0.0 x 4, 6x ] nonadecane (nonadec) -7-en-18-yl ester.

Other HCV NS3 and NS3/4A protease inhibitors include "boceprevir" or "SCH-503034": (1R,5S) -N- [ 3-amino-1 (cyclobutylmethyl) -2, 3-dioxopropyl ] -3- [2(S) - [ [ [ (1, 1-dimethylethyl) amino ] carbonyl ] amino ] -3, 3-dimethyl-1-oxobutyl ] -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane- (S) -carboxamide; and "telaprevir" or "VX-950": (1S,3aR,6aS) -2- [ (2S) -2- [ [ (2S) -cyclohexyl [ (pyrazinylcarbonyl) amino ] acetyl ] amino ] -3, 3-dimethylbutyryl ] -N- [ (1S) -1- [ (cyclopropylamino) oxyacetyl ] butyl ] octahydrocyclopenta [ c ] pyrrole-1-carboxamide; "vaniprevir" or "MK-7009": (1R,21S,24S) -21-tert-butyl-N- ((1R,2R) -1- { [ (cyclopropylsulfonyl) amino ] carbonyl } -2-ethylcyclopropyl) -16, 16-dimethyl-3, 19, 22-trioxa-2, 18-dioxa-4, 20, 23-triazatetracyclo [21.2.1.1.0] heptacosan-6, 8, 10-triene-24-carboxamide; "MK-5172 (1aR,5S,8S,10R,22aR) -5- (1, 1-dimethylethyl) -1,1a,3,4,5,6,9,10,18,19,20,21,22 a-tetradecahydro-14-methoxy-3, 6-dioxo-8H-7, 10-methylenecyclopropane [18,19] [1,10,3,6] dioxadiazacyclononadeno [11,12-b ] quinoxaline-8-carboxylic acid; "TMC-435": (2R,13aR,14aR,16aS, Z) -N- (cyclopropylsulfonyl) -2- (2- (4-isopropylthiazol-2-yl) -7-methoxy-8-methylquinolin-4-yloxy) -6-methyl-5, 16-dioxo-1, 2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16 a-hexahydro (hexadecahydro) cyclopropane [ g ] pyrrole [1,2-c ] [1,3,6] triazacyclodecyne-14 a-carboxamide; and "narlaprevir" or "SCH-900518": (1R,2S,5S) -3- ((S) -2- (3- (1- (tert-butylsulfonylmethyl) cyclohexyl) ureido) -3, 3-dimethylbutyryl) -N- ((S) -1- (cyclopropylamino) -1, 2-dioxohept-3-yl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide.

The term "triple therapy treatment" refers to a treatment regimen for a patient infected with HCV that comprises peginterferon, ribavirin and one or more direct acting antiviral agents. "direct acting antiviral agents" exert specific antiviral effects independent of immune function. Examples of direct acting antiviral agents for HCV include, but are not limited to, NS3/4A protease inhibitors, NS5B polymerase inhibitors, NS5A inhibitors, IRES inhibitors, and helicase inhibitors. One example of a triple therapy treatment includes peginterferon, ribavirin, and an hcv ns5B polymerase inhibitor. Another example of a triple therapy treatment comprises peginterferon, ribavirin and an HCV NS3/4A protease inhibitor. Other examples of triple therapy treatments include peginterferon, ribavirin, hcv ns5B polymerase inhibitors, and hcv ns3/4A protease inhibitors (which may also be referred to as "quadruple therapy treatments").

The first line of treatment (referred to as "standard of care" or "SOC") currently recommended for patients with Chronic Hepatitis C (CHC) is a 48 week "duration" combination of pegylated interferon alfa with ribavirin in patients carrying genotype1 or 4 virus, and a 24 week duration in patients carrying genotype 2 or 3 virus. SOC that has been proposed for the first treatment of patients with chronic hepatitis C genotype1 virus infection in some countries (e.g., members of the United states and European Union) is currently the combination therapy of HCV protease inhibitors (boceprevir or telaprevir) with pegylated interferon alpha (PEG-IFN) plus Ribavirin (RBV) (GhanyMG et al, "adaptive treatment of genotype1Chronic hepatitis C virus infection Infection:2011 practical Guideetienbythe American Association for study of liver disease 2011: 1433-. However, the term "SOC" as used in this document refers to treatment with PEG-IFN plus RBV alone.

Combination therapy with ribavirin is found to be more effective than interferon alpha monotherapy in patients who relapse after one or more courses of interferon alpha therapy, as well as in previously untreated patients. However, ribavirin exhibits significant side effects, including teratogenicity and carcinogenicity. Furthermore, ribavirin causes hemolytic anemia and requires dose reduction or discontinuation of ribavirin therapy in about 10-20% of patients, which may be associated with accumulation of ribavirin triphosphate in erythrocytes. Therefore, in order to reduce the cost of treatment and the occurrence of adverse events, it is desirable to adjust the treatment to a shorter duration without compromising efficacy.

The terms "allele" and "allelic variant" as used herein refer to alternative forms of a gene, including introns, exons, intron/exon junctions, and 3 'and/or 5' untranslated regions associated with the gene or portions thereof. Typically, alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a particular gene may differ from one another in a single nucleotide or in several nucleotides, and may include substitutions, deletions, and insertions of nucleotides.

The term "polymorphism" as used herein refers to the coexistence of more than one form of a nucleic acid (e.g., allelic variants thereof). Portions of a gene having at least two different forms, i.e., two different nucleotide sequences, are referred to as polymorphic regions of the gene. A polymorphic region may be a single nucleotide, i.e., a "single nucleotide polymorphism" or "SNP," the identity of which differs in different alleles. The polymorphic region may also be several nucleotides in length.

Various methods of detecting polymorphisms are known and may be used in conjunction with the present invention. Generally, these methods include identifying one or more mutations in the underlying nucleic acid sequence, either directly (e.g., allele-specific PCR) or indirectly (identifying changes to a second molecule, such as a protein sequence).

One well-known method for genotyping single nucleotide polymorphisms is the use of IlluminaSNP bead chips, in which fragmented and denatured DNA samples are hybridized to corresponding 50-mer oligonucleotides attached to beads on the chip. After hybridization, the chip was processed for enzymatic single base extension followed by fluorescent staining and imaging. Finally, the fluorescence intensity was detected and used for SNP genotyping (genotyping).

The single nucleotide polymorphisms "rs 12979860", "rs 12980275" and "rs 8099917" refer to SNPs identified by their accession numbers in the SNP database (dbSNP, www.ncbi.nlm.nih.gov/SNP /) and are localized in multiple regions around the human chromosome 19 IL28b gene.

The single nucleotide polymorphism "rs 12148487" refers to the SNP identified by its accession number in dbSNP and is located in the intronic region of the human chromosome 15 SLCO3a1 gene, which is a member of the solute carrier organic anion transporter family. Synonymous terms for SLCO3A1 include OATP-D, OATP3A1.OATPD and SLC21A 11.

Examples

Introduction to

The HCV protease encoded by the non-structural gene 3/4A (NS3/4A) represents a clinically validated anti-HCV target and is critical for post-translational modification of HCV polypeptides and viral replication. Danoprevir (also known as RO5190591, RG7227 or ITMN-191) is a macrocyclic peptidomimetic compound that competitively inhibits the hcv ns3/4A protease. Due to the high antiviral potential, specificity and safety profile of Danoprevir, Danoprevir was chosen for the development of oral agents for the treatment of patients with chronic hcv (chc).

Design of research

Study NV21075 is a randomized, double-blind, multicenter phase 2 clinical study to evaluate the efficacy and safety of a combination of danoprevir with peginterferon alfa 2a and ribavirin (triple therapy combination) compared to the currently approved peginterferon alfa 2a and ribavirin combination (standard of care or SOC) at 12 weeks of dosing in the first treatment of patients infected with chronic hepatitis c genotype 1. Patients enrolled in study NV21075 were randomized to one of four treatment groups (figure 1) (about 60 patients in groups a-C and about 30 patients in group D). In the response-directed treatment group (described below), patients with extended RVR (edrvr, defined as failure to detect hcv rna from weeks 4-20) discontinued all therapy at week 24.

Treatment group A, B, C: patients received double-blind experimental treatment with danoprevir300mg tid (group a), 600mg bid (group B) or 900mg bid (group C) and standard of care treatment (SOC, Pegasys180gscqw + Copegus1000mg (<75kg) or 1200mg (. Patients who achieved RVR (defined as undetectable hcv rna by week 4) and remained undetectable by week 22 (i.e., achieved RVR) discontinued all treatment by week 24. Patients who did not achieve edrvr received SOC for an additional 24 weeks with a total treatment duration of 48 weeks. Treatment group D: patients received double-blind placebo treatment for 12 weeks followed by SOC36 weeks for a total duration of 48 weeks.

RCR patient population using IL28B genotyping and Whole genome Association study (GWAS) Body

The Roche Clinical Repository (RCR) is an optional process requiring separate consentSequencing; data is anonymized to ensure patient privacy. Use ofReal-time PCR, using RCRDNA samples, determined the IL28B status relative to rs 12979860. Summarizing the two subgroups, efficacy endpoints of CC versus non-CC (CT and TT). For the NV21075 study, genotyping data was obtained from 171 patient samples. In addition, these RCRDNA samples were used for GWAS and genotyped by Illumina HumanOmni ExpressBeadchip using a total of 733,202 Single Nucleotide Polymorphisms (SNPs). 725,947SNPs in 733,202 were passed through Quality Control (QC). Genome-wide association analysis was performed in 166 patients with a complete clinical data set.

Results

For intermittent analysis of NV21075, by having a virological response (e.g., by Roche)AmpliPrep/HCVTestv1.0 assay failed to detect HCVRNA [ detection limit 15IU/mL]) The percentage of patients in (c) determines the efficacy during the first 12 weeks of treatment. If the patient's data is lost (which may be due to missed assessments or outside the allowed visit window, or early exit from treatment, missed follow-up assessments), the patient is considered to be a non-responder at the time of the planned visit.

Preliminary results indicate that 88.1%, 89.2% and 92.0% of patients in treatment groups A, B and C, respectively, had undetectable hcv rna levels at the end of 12 weeks of treatment compared to 43.3% of patients receiving SOC (fig. 2). Patients treated with Danoprevir experienced a sharp drop in mean hcv rna levels within the first 2-4 weeks of treatment and remained lower until week 12 compared to patients receiving SOC (figure 3).

For a sample of 171 patients, in which genotyping data was available for the rs12979860SNP in the IL28B gene, 56 patients had a CC (therapeutically favorable) genotype, while 115 patients carried a non-CC (therapeutically unfavorable) genotype. Regardless of the treatment group they were in, CC patients showed better virological response rates than non-CC patients, especially within the first two weeks of treatment (fig. 4).

In a subgroup of patients from treatment groups a and B, whose hcv rna levels were available within 12 weeks of post-treatment follow-up (i.e., 12 weeks after treatment discontinuation), correlations were determined between those patients who achieved early response measures, RVR2, RVR4, eRVR and those who achieved SVR-12. Of the 68 patients achieving RVR2 in treatment groups A and B, 64 patients achieved SVR-12, and RVR2 produced 94% Positive Predictive Value (PPV) for SVR-12. In comparison, of the 97 patients achieving RVR4, 87 patients achieved SVR-12, and RVR4 produced 90% PPV for SVR-12. PPV for eVR on SVR-12 is also 90%, since 84 of the 93 patients who achieved eRVR also achieved SVR-12. These results show that early response measures, especially RVR2, are highly relevant to SVR-12.

To identify additional single nucleotide polymorphisms that may be associated with clinical efficacy endpoints in response to either dinoprevir plus SOC triple therapy or SOC-only therapy in HCV treatment, patient samples were genotyped on the illumina omniexpress chip set. After quality control, 725,947SNPs with genotype recognition were generated. Logistic regression models were used to test the association between individual SNPs and a rapid virological response-2 weeks (RVR2) after adjustment of viral load and sample ethnicity at baseline. Not surprisingly, the rs12979860SNP showed significant association with RVR2 with a p-value of 6x10-8 (fig. 5A). After adjustment of rs12979860, an additional SNP with p <10-5 on chromosome 15 was observed (fig. 5B), the most significant of which was rs12148487, located in the intron of the gene encoding the solute carrier organic anion transporter, SLCO3a 1. In the NV21075 population, an association between RVR2 and rs12148487 minor allele a was observed with an Odds Ratio (OR) higher than 21(OR 21.87, 95% CI [ 6.30; 96.88], p 5.6x 10-8). These results were obtained using a dominant model in logistic regression against rs12148487, with AA and GA genotypes designated as '1' and GG genotype as '0'. Fig. 6 shows a bar chart of the association between RVR2 and rs 12148487.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Reference to the literature

1.Lavanchy,D.GlobalSurveillanceandControlofHepatitisC:ReportofaWHOConsultation.JViralHepatitis.1999；6:35-47.

2.ConsensusStatement:EASL/InternationalConsensusConferenceonHepatitisC.JHepatol.1999；31(Suppl1):38.

3.RecommendationsfromtheNationalInstitutesofHealthConsensusDevelopmentConferenceStatement:ManagementofHepatitisC:2002.HepatologyNov2002；Vol36No5:1039.

FriedMW, ShiffmanML, ReddyKR, SmithC, MarinosG, GoncalesFLJr, et al 2002.Peginterferonalfa-2 aplusrivivirinforchronicpapatis Cvirus. NEJMSepember 262002; vol347No13:975-

Hadziyannnisis SJ, SetteJr. H, MorganTR et al, pegprevented feronaalfa-2 aandribarivinino thermocondustrin thermocondustric thermocondustria C.AnnIntMed2004Vol140:346-355.

6.MannsMP,McHutchisonJG,GordonSC,RustgiVK,ShiffmanM,ReindollarR,GoodmanZD,KouryK,LingM,AlbrechtJK.Peginterferonalfa-2bplusribavirincomparedwithinterferonalfa-2bplusribavirinforinitialtreatmentofchronichepatitisC:arandomisedtrial.Lancet2001；358:958-965.

7.ReportNo.1022744；2006:InhibitionofRNAsynthesisbyby？-D-2-deoxy-2-fluoro-2-C-methylcytidine(RO4995855)intheHCVSubgenomicRepliconSystem.RochePaloAltoLCC.

8.RO5024048InvestigatorsBrochure,November2008.

9.PEG-IFNalfa-2aInvestigatorsBrochure,July,2006.

10.PEGASYSCoreDataSheetVersion1.4,January19,2005.

11.SidwellRW,RobinsRK,andHillyardIW.Ribavirin:anantiviralagent.PharmacolTher1979；6:123-146.

12.GilbertBEandKnightV.Minireview:Biochemistryandclinicalapplicationsforribavirin.AntimicrobAgentsChemother.1986；30:201-205.

Dusheiko, MainJ, Thomash et al Ribavirentreatentrentfor PatientswitchchronicpapitsC Results of Aplacebo-controllerdtrial.JHepatol1996; 25:591-598.

DiBisceglieAM, ConjeevaramHS, FriedMW et al, rivarinatherotherapeutic for phonocatheterephatitis c. arandomized, double-blind, placebo-controllerdtrial. annlint med1995; 123:897-903.

15.ConjeevaramHS,FriedMW,JeffersLJ,TerraultNA,Wiley-LucasTE,AfdhalN,BrownRS,BelleSH,HoofnagleJH,KleinerDE,HowellCD.PeginterferonandribavirintreatmentinAfricanAmericanandCaucasianAmericanpatientswithhepatitisCgenotype1.Gastroenterology2006；131:470-477.

16.DienstagJL,McHutchisonJG.AmericanGastroenterologicalAssociationtechnicalreviewonthemanagementofhepatitisC.Gastroenterology2006；130:231-264.

17.MissihaS,HeathcoteJ,ArenovichT,KhanK.Impactofasianraceonresponsetocombinationtherapywithpeginterferonalfa-2aandribavirininchronichepatitisC.AmJGastroenterol2007；102:2181-2188.

18.ReddyKR,MessingerD,PopescuM,HadziyannisSJ.Peginterferonalpha-2a(40kDa)andribavirin:comparableratesofsustainedvirologicalresponseinsub-setsofolderandyoungerHCVgenotype1patients.JViralHepat2009；16:724-731.

19.FerenciP,FriedMW,ShiffmanML,SmithCI,MarinosG,GoncalesFL,Jr.,HaussingerD,DiagoM,CarosiG,DhumeauxD,CraxiA,ChaneacM,ReddyKR.PredictingsustainedvirologicalresponsesinchronichepatitisCpatientstreatedwithpeginterferonalfa-2a(40KD)/ribavirin.JHepatol2005；43:425-433.

20.Martinot-PeignouxM,MaylinS,MoucariR,RipaultMP,BoyerN,CardosoAC,GiuilyN,CastelnauC,PouteauM,SternC,AuperinA,BedossaP,AsselahT,MarcellinP.Virologicalresponseat4weekstopredictoutcomeofhepatitisCtreatmentwithpegylatedinterferonandribavirin.AntivirTher2009；14:501-511.

21.AsselahT,BiecheI,SabbaghA,BedossaP,MoreauR,VallaD,VidaudM,MarcellinP.GeneexpressionandhepatitisCvirusinfection.Gut2009；58:846-858.

22.GeD,FellayJ,ThompsonAJ,SimonJS,ShiannaKV,UrbanTJ,HeinzenEL,QiuP,BertelsenAH,MuirAJ,SulkowskiM,McHutchisonJG,GoldsteinDB.GeneticvariationinIL28BpredictshepatitisCtreatment-inducedviralclearance.Nature2009；461:399-401.

23.SuppiahV,MoldovanM,AhlenstielG,BergT,WeltmanM,AbateML,BassendineM,SpenglerU,DoreGJ,PowellE,RiordanS,SheridanD,SmedileA,FragomeliV,MullerT,BahloM,StewartGJ,BoothDR,GeorgeJ.IL28BisassociatedwithresponsetochronichepatitisCinterferon-alphaandribavirintherapy.NatGenet2009；41:1100-1104.

24.TanakaY,NishidaN,SugiyamaM,KurosakiM,MatsuuraK,SakamotoN,NakagawaM,KorenagaM,HinoK,HigeS,ItoY,MitaE,TanakaE,MochidaS,MurawakiY,HondaM,SakaiA,HiasaY,NishiguchiS,KoikeA,SakaidaI,ImamuraM,ItoK,YanoK,MasakiN,SugauchiF,IzumiN,TokunagaK,MizokamiM.Genome-wideassociationofIL28Bwithresponsetopegylatedinterferon-alphaandribavirintherapyforchronichepatitisC.NatGenet2009；41:1105-1109.

Claims (5)

1. Use of an agent that identifies a nucleotide present at the single nucleotide polymorphism rs12148487 in the manufacture of a diagnostic agent for predicting the response of a human subject infected with HCV to treatment with peginterferon alfa-2a, ribavirin and a direct acting antiviral agent, wherein the presence of at least one a allele at rs12148487 in said subject indicates a higher probability that said subject will achieve a rapid virological response (RVR2) within two weeks to said treatment relative to a subject having two G alleles present at rs 12148487.

2. The use of claim 1, wherein said subject is infected with HCV genotype-1 or HCV genotype-4.

3. The use of claim 2, wherein said direct acting antiviral agent is an HCV protease inhibitor.

4. The use of claim 3, wherein said HCV protease inhibitor is selected from the group consisting of danoprevir, boceprevir, telaprevir, vaniprevir, MK-5172, TMC-435, and narloprevir.

5. The use of claim 4, wherein said HCV protease inhibitor is danoprevir.