UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)

UvA-DARE (Digital Academic Repository)

MicroRNA’s in chronic hepatitis B and C virus infection

van der Ree, M.H.

Link to publication

LicenseOther

Citation for published version (APA):van der Ree, M. H. (2017). MicroRNA’s in chronic hepatitis B and C virus infection.

General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.

Download date: 24 Feb 2021

CHAPTER 2MIRAVIRSEN DOSING IN CHRONIC HEPATITIS C PATIENTS RESULTS IN

DECREASED MICRORNA-122 LEVEL WITHOUT AFFECTING OTHER

MICRORNA’S IN PLASMA

Meike H. van der Ree, Adriaan J. van der Meer, Ad C. van Nuenen, Joep de Bruijne, Søren Ottosen, Harry L. Janssen,

Neeltje A. Kootstra §, Hendrik W. Reesink §

§ Both authors contributed equally to this work

Alimentary Pharmacology and Therapeutics 2016;43:102-13

CHAPTER 2

32

SUMMARYBackground: MicroRNA-122 (miR-122) is an important host factor for hepatitis C virus

replication. Administration of miravirsen, an anti-miR-122 oligonucleotide, resulted

in a dose dependent and prolonged decrease in HCV RNA levels in chronic hepatitis

C patients.

Aim: To assess the plasma level of various miRNAs in patients dosed with miravirsen.

Methods: We included 16 of 36 chronic hepatitis C patients who received five injections

of either 3 mg/kg (n = 4), 5 mg/kg (n = 4), 7 mg/kg (n = 4) miravirsen or placebo (n = 4)

over a 4-week period in a double-blind, randomised phase 2a study. Plasma levels of 179

miRNAs were determined by qPCR and compared between patients dosed with miravirsen

or placebo.

Results: Median plasma miR-122 level at baseline in patients receiving miravirsen was

3.9 x 103 compared to 1.3 x 104 copies/4 μl in placebo-dosed patients (P=0.68). At week

1, 4, 6 and 10/12, patients dosed with miravirsen had respectively a median 72-fold,

174-fold, 1109-fold and 552-fold lower expression of miR-122 than at baseline (P=0.001,

as compared to patients receiving placebo). At week 4 of dosing, miRNA-profiling

demonstrated a significant lower expression of miR-210 and miR-532-5p compared to

baseline (3.0 and 4.7-fold lower respectively). However, subsequent longitudinal analysis

showed no significant differences in miR-210 and miR-532-5p plasma levels throughout

the study period.

Conclusions: We demonstrated a substantial and prolonged decrease in plasma miR-122

levels in patients dosed with miravirsen. Plasma levels of other miRNAs were not

significantly affected by antagonising miR-122.

PLASMA MIRNA-LEVELS FOLLOWING MIRAVIRSEN DOSING

33

2

INTRODUCTIONMicroRNAs (miRNAs) are small (~22 nucleotides), noncoding RNA molecules involved in

various cellular processes 1,2. MiRNAs regulate gene expression at a posttranscriptional

level by binding to the 3’ untranslated region (UTR) of target messenger RNA (mRNA) 3,4.

The role of miRNAs has been extensively studied since their discovery approximately

20 years ago 5,6. MiRNAs are now recognised as important players in the pathogenesis

of a number of liver diseases, and are promising biomarkers and targets for

therapeutic intervention 7,8.

Hepatitis C virus (HCV) is an envelope, single-stranded RNA virus of the family Flaviviridae.

The HCV genome contains a single open reading frame with a length of 9.6-kb which is

translated into a polyprotein of about 3000 amino acids 9. The HCV genome is flanked by

the 5’ and 3’ UTR which are important for the translation and replication of the viral RNA.

It is known that HCV uses host cellular miRNAs during its replicative cycle and is able to

modulate the expression of cellular miRNAs to favour viral persistence 10. MicroRNA-122

(miR-122) is a liver-specific miRNA that binds to the 5’ UTR of the HCV genome to promote

HCV RNA stability and accumulation 11,12. The miR-122-HCV complex protects the HCV

genome from degradation by cellular exonucleases Xrn1/Xrn2 13,14, and is believed to

prevent the induction of an innate immune response 11,15. MiR-122 is also involved in

the regulation of lipid metabolism and may act as a tumour suppressor 16–18.

The majority of HCV infected patients develop a chronic hepatitis C (CHC) virus infection

and are at risk to develop cirrhosis, leading to clinical complications such as hepatocellular

carcinoma (HCC) 19,20. The aim of CHC treatment is to achieve a sustained virological

response (SVR), which is associated with a reduced occurrence of liver failure and HCC,

and with prolonged overall survival 21–23. The recent development and registration of many

highly potent direct-acting anti-viral drugs (DAA) have resulted in high SVR rates following

HCV treatment. Most DAA’s target viral proteins, such as NS3 protease inhibitors, NS5B

polymerase inhibitors and NS5A replication inhibitors whereas other drugs target host

factors that are essential for HCV replication, such as miR-122 24. In 2011-2012, a phase 2a

study was performed in CHC patients infected with HCV genotype 1 who received 5-weekly

injections of miravirsen, a locked nucleic acid-modified phosphorothiate oligonucleotide

targeting miR-122, which resulted in a prolonged and dose-dependent decrease in HCV

RNA levels 24. HCV RNA levels became undetectable in five patients, but levels of the virus

eventually rebounded in all patients 24,25. A C3U nucleotide change was observed in

the HCV 5’UTR in patients with a viral rebound after miravirsen dosing 26. Interestingly,

a wide variability in patient responses to miravirsen was observed suggesting that host

and/or viral factors may influence treatment response. It is unknown how many miR-122

copies are required for HCV replication and whether miravirsen is able to sequester

all hepatic miR-122. Furthermore, it is unknown how long the inhibition of miR-122 is

CHAPTER 2

34

preserved after miravirsen dosing. In addition, the direct or indirect effect of miR-122

inhibition on the expression level of other miRNAs has never been established.

Therefore, the primary objective of this study was to assess changes in plasma miR-122

levels in CHC patients before, during and after miravirsen dosing. The secondary objective

was to assess if inhibiting miR-122 alters the expression levels of other miRNAs.

MATERIALS AND METHODSStudy populationWe have used plasma samples of CHC patients who participated in a previous multicenter,

double-blind, phase 2a study in which patients were randomised in a 3:1 ratio to receive

either miravirsen (3, 5 or 7 mg/kg) or placebo (ClinicalTrials.gov number, NCT01200420). 24

These plasma samples were only collected in patients who were recruited at one of

the Dutch study sites (Academic Medical Center, Amsterdam or Erasmus Medical Center,

Rotterdam) (n = 17). Of these 17 patients, the 16 patients included in this study received

3 mg/kg (n = 4), 5 mg/kg (n = 4) or 7 mg/kg (n = 4) miravirsen, or placebo (n = 4). One

placebo-dosed patient was excluded, to achieve an equal number or patients in each dose

group. All patients were treatment naïve, and infected with HCV genotype 1. Miravirsen

(or placebo) was administered subcutaneously in 5-weekly doses over a 29-day period.

After miravirsen dosing, patients returned for follow-up visits over a period of 14 weeks.

The additional blood samples were collected at baseline, week 1, week 4, week 6 and

week 10 or 12. The study was carried out in compliance with the protocol, the principles

laid down in the Declaration of Helsinki, in accordance with the ICH Harmonised Tripartite

Guideline for Good Clinical Practice and the local national laws governing the conduct of

clinical research studies.

Biochemical and virological testsSeveral biochemical and virological analyses were conducted in the phase 2a study,

including measurement of alanine aminotransferase (ALT) and HCV RNA levels. HCV RNA

levels were measured using the Abbott Real-Time HCV assay, with a reported lower limit

of detection and quantification of 12 IU per mL.

Current study designThis study comprised three steps: (i) Absolute and relative quantification of plasma

miR-122 levels in CHC patients dosed with miravirsen (n = 12) and placebo (n = 4) at

baseline, week 1, 4, 6 and 10/12; (ii) Assessment if antagonising miR-122 results in

changes in expression levels of other miRNAs. Therefore, a miRNA profile was determined

to evaluate the difference in plasma miRNA levels at week 4 compared to baseline in

CHC patients dosed with miravirsen or placebo (fold change level); (3) Analysis of

PLASMA MIRNA-LEVELS FOLLOWING MIRAVIRSEN DOSING

35

2

the differential expression of miRNAs identified in the miRNA-profile in longitudinally

obtained plasma samples from patients dosed with miravirsen or placebo. MiRNAs were

selected for the longitudinal analysis if they fulfilled the following criteria; a fold change

≥2 or ≤0.5 (log10 ≥ 0.301 or ≤-0.301) in patients dosed with miravirsen and a fold change

level between 0.5 and 2 (log10 -0.301 to 0.301) in placebo dosed patients, and a significant

difference (P < 0.05) in fold change level between miravirsen and placebo dosed patients.

RNA extraction, cDNA synthesis and qPCRTotal RNA was extracted from 200 μl plasma using the miRCURY RNA isolation kit (Exiqon,

Vedbæk, Denmark) according to the manufacturer’s protocol. Synthetic cel-miR-39 (Qiagen,

Hilden, Germany) was spiked into the lysis buffer to check RNA isolation efficiency. RNA

samples were stored at –80 °C. cDNA was synthesised using microRNA cDNA synthesis

kit (Quanta BioSciences, Gaithersburg, MD, USA) and miRCURY LNA Universal RT cDNA

synthesis Kit (Exiqon, Denmark) (Data S1). An individual miR-122 qPCR assay was used to

determine plasma miR-122 levels (Data S1). A serum/plasma focus microRNA qPCR panel

(Exiqon, Denmark) was used to determine the expression level of 179 different miRNAs

that are known to be found in plasma (Data S1).

Data analysis and statisticsThe amplification curves of miRNAs were analysed using the Lightcycler 480 software

(Roche Diagnostics, Ltd, Rotkreuz, Switzerland) for the quantification of cycles (Cq) and

for the melting curve analysis. All assays were inspected for distinct melting curves. Data

were pre-processed using GenEx pro software (MultiD Analyses, Goteborg, Sweden)

(Data S1). Suitable reference genes were found by applying the NormFinder and GeNorm

algorithms on the miRNA profile panel results 27,28. All miRNAs were normalised using

the ∆Cq method (= Cq miRNA of interest – Cq reference miRNA). The normalised

individual data points are presented as the log10 2 - ∆Cq (Table S2) 29. The difference

in the expression level of miRNAs between baseline and either week 1, 4, 6 or 10/12

was calculated with the comparative Cq-method (= 2 ^ - (Cq miRNa of interest – Cq reference) baseline

- (Cq miRNa of interest – Cq reference) week x) and expressed as the fold change level 29. Formulas and

interpretation of the delta Cq and comparative delta Cq method are described in Table S2.

We tested for difference in plasma miR-122 levels between treatment groups using

the Mann–Whitney U-test, and one-way ANOVA with post-test for linear trend using

SPSS (IBM SPSS Statistics for Windows, Version 22.0, Armonk, NY, USA.) and GraphPad

Software, La Jolla, CA. (version 5). Correlations were analysed using the Spearman’s

rank correlation coefficient using SPSS (version 22.0). We tested for difference in miRNA

profiling data between treatment groups using t-test and corrected for multiple testing

(Benjamini–Hochberg method). Unsupervised hierarchical clustering was performed with

GenePattern software.

CHAPTER 2

36

Table 1. Baseline patient characteristics

Miravirsen

Placebo3 mg/kg 5 mg/kg 7 mg/kg

Patients, n 4 4 4 4Median age, years (range) 56 (46 - 66) 49 (33 - 65) 42 (35 - 51) 54 (47 - 66)Male/Female 2/2 3/1 3/1 1/3RaceWhite 4 4 3 4Asian 0 0 1 0HCV RNA – log10 IU/ml 6.3 ± 0.6 6.2 ± 0.3 5.6 ± 0.5 6.5 ± 0.1HCV subtype1a 3 2 3 31b 1 1 1 11a/3a 0 1 0 0ALT level – U/L 70 ± 38 77 ± 18 120 ± 89 89 ± 56Plasma miR-122 level – log10

copies/4 µl

3.5 (2.7 - 3.7) 3.6 (2.7 - 4.3) 2.8 (0.7 – 4.1) 3.0 (2.7 – 4.4)

Data are given as median (minimum–maximum), as mean ± SD or as number. ALT, alanine aminotransferase.

RESULTSPatient characteristicsThis study included 16 patients of whom 12 patients received various doses of miravirsen

and 4 received placebo 24. Baseline characteristics levels were similar in the different study

groups (Table 1).

Absolute plasma miR-122 levels at baseline and after miravirsen dosingThe median plasma miR-122 level at baseline in patients receiving miravirsen was 3.9 x

103 compared to 1.3 x 104 copies/4 μl in placebo treated patients (P = 0.68) (Figure 1).

One week after the first dose, the median plasma miR-122 level was significantly lower

in patients dosed with miravirsen than in those who received placebo, respectively 3.1 x

101 versus 1.1 x 104 copies/4 μl (P = 0.001) (Figure 1). Median plasma levels of miR-122

remained low in patients administered with miravirsen compared to those administered

with placebo throughout the study period (P = 0.001) (Figure 1).

Fold change in plasma miR-122 levels upon miravirsen treatmentOne week after receiving the first dose of miravirsen, plasma miR-122 levels were 72-fold

lower than at baseline in patients dosed with miravirsen, as compared to a 1.1-fold

higher miR-122 plasma level in placebo dosed patients (P = 0.001). Patients dosed with

miravirsen had a median 174-fold, 1109-fold and 552-fold lower expression of miR-122

compared to baseline at week 4, 6, and 10/12, compared to a 1.5-fold higher, 5.8-fold

PLASMA MIRNA-LEVELS FOLLOWING MIRAVIRSEN DOSING

37

2

Figure 1. Absolute plasma miR-122 level. Absolute plasma levels of miR-122 in patients dosed with miravirsen compared to placebo. The median absolute level of miR-122 at baseline was similar between patients dosed with placebo and miravirsen. At week 1, 4, 6, and 10/12 median absolute miR-122 plasma levels were lower in patients dosed with miravirsen, as compared to placebo. The boxes represent the median and range, and Mann-Whitney-U test was used to compare the different treatment groups. LLOD is lower limit of detection.

lower and 3.1-fold lower expression of miR-122 in patients dosed with placebo, P-values all

P = 0.001. There was a significant linear trend for fold change in plasma miR-122 levels and

miravirsen dose at week 1, 4, 6 and 10/12 (P < 0.001) (Figure 2A). All patients receiving

miravirsen showed an initial decrease in plasma miR-122 levels (Figure 2). At week 10/12,

all patients dosed with 3 and 5 mg/kg miravirsen had stable or increasing relative plasma

miR-122 levels from nadir, whereas relative plasma miR-122 levels of 3/4 patients dosed

with 7 mg/kg were still decreasing (Figure 2B).

CHAPTER 2

38

Association of plasma miR-122 levels with clinical parametersThe mean HCV RNA level at baseline for patients dosed with miravirsen was 6.1 log10 IU/

mL, and the viral load decreased significantly to 5.3 at week 4 (P = 0.01), 4.8 (P = 0.005) at

week 6 and 4.7 (P = 0.008) log10 IU/mL at week 10/12. At baseline, there was no correlation

between absolute plasma miR-122 and HCV RNA levels (R = 0.29, p = 0.28) (Figure 3A). No

correlation was found between the change in HCV RNA level and fold change in plasma

miR-122 level at week 4 (R = 0.42, P = 0.10) (Figure 3B).

Mean ALT levels at baseline were similar in patients dosed with miravirsen and placebo

(Table 1), and showed no significant correlation with absolute plasma miR-122 levels

(Figure 3C). ALT levels decreased to 44 U/L at week 4 and onwards in anti-miR-122 dosed

patients compared to baseline (P = 0.008). At week 4, fold change in plasma miR-122 level

showed a significant correlation with the changes in ALT levels (log10 U/L) (R = 0.57, P =

0.02) (Figure 3D).

MiRNA profiling: change in plasma miRNA levels upon miravirsen dosingAt baseline and week 4, plasma levels of 179 miRNAs were analysed of which 13 miRNAs

were eliminated from further analyses because of a too low number of samples with

valid data (Figure 4A). Among the remaining 166 miRNAs, we identified 19 miRNAs with

a fold change level ≥2 or ≤0.5 in patients dosed with miravirsen and a fold change level

between 0.5 and 2 in placebo-dosed patients (Figure 4A). Unsupervised hierarchical

clustering using these 19 miRNAs clustered all patients dosed with miravirsen or placebo

in separate groups (Figure 4B). Next to miR-122, two miRNAs (miR-210 and miR-532-5p)

were differentially expressed between placebo and miravirsen dosed patients with a P <

0.05, and were selected for longitudinal analysis (Figure 4A, 4B).

At baseline, the plasma level of miR-122 and miR-210 were comparable between miravirsen

and placebo dosed patients, with a mean delta Cq value of respectively 0.80 versus 0.64

(P = 0.58) and -0.79 vs. -0.80 (P = 0.98) (Table 2 and Figure S1). The mean delta Cq value

of miR-532-5p at baseline was higher in patients who received miravirsen compared to

placebo, respectively 3.65 vs. 2.70, P = 0.03 (Table 2 and Figure S1). At week 4, patients

dosed with miravirsen had a mean 3.0-fold lower expression of miR-210 than at baseline,

as compared with a 1.5-fold higher expression in placebo dosed patients (P = 0.007)

(Table 2). MiR-532-5p levels were 4.7-fold lower compared to baseline, whilst placebo-

dosed patients showed a mean 1.2-fold increase in plasma miR-532-5p levels

(P = 0.02) (Table 2).

Longitudinal analysis of miRNAs identified in miRNA-profilingPlasma levels of miR-210 and miR-532-5p were measured at baseline, week 1, 4, 6 and

10/12 in an individual RT-qPCR assay. In patients dosed with miravirsen, plasma miR-210

PLASMA MIRNA-LEVELS FOLLOWING MIRAVIRSEN DOSING

39

2

Figure 2. Fold change in plasma miR-122 level compared to baseline. (A) Fold change in relative plasma miR-122 levels per dose group per week, compared to baseline. The median fold decrease in miR-122 level at week 1, 4 ,6 and 10/12 was lower in patients dosed with miravirsen, as compared to placebo. A significant linear trend, indicated by the black line, was found for fold change in plasma miR-122 levels and miravirsen dose. The boxes represent the range. * P <0.05; ** P <0.01; *** P <0.001. *** above the black line indicates a significant linear trend with P = 0.001. Mann-Whitney-U test and one-way ANOVA with a post test for linear trend was used to compare different study groups. (B) Individual plots of fold change levels of plasma miR-122 in all patients.

A)

B)

CHAPTER 2

40

A) B)

C) D)

Figure 3. Correlation plasma miR-122 with HCV RNA and ALT level. Correlation between baseline absolute plasma level of miR-122 and (A) HCV RNA level, and (B) ALT level. Correlation between week 4 fold change miR-122 levels and (C) HCV RNA level, and (D) ALT levels. The correlation coefficient (R) is calculated using the Spearman’s correlation test. The 95% confidence interval is shown.

levels showed a mean 1.5-fold increase at week 1 (P = 0.69), 1.5-decrease at week 4 (P

= 0.12), 2.6-fold decrease at week 6 (P = 0.79) and 1.1-fold decrease at week 10/12 (P =

0.48), as compared to placebo (Table 2 and Figure 5A). In patients dosed with miravirsen

vs. placebo, plasma miR-532-5p levels showed a median 2.0-fold versus 1.0-fold increase

between baseline and week 1 (P = 0.55), 1.4-fold vs. 1.7-fold increase at week 4 (P = 0.84),

7.6-fold vs. 3.0-fold decrease at week 6 (P = 0.42) and 3.2-fold vs. 3.1-fold decrease at

week 10/12 (P = 0.98) (Table 2 and Figure 5B).

DISCUSSIONThis is the first study to assess changes in plasma miRNA levels in CHC patients dosed with

an anti-miR-122 oligonucleotide. We demonstrate a substantial and prolonged decrease

in plasma miR-122 levels in patients dosed with miravirsen. Furthermore, plasma levels

of other miRNAs were shown to be variable in both patients dosed with placebo and

PLASMA MIRNA-LEVELS FOLLOWING MIRAVIRSEN DOSING

41

2

Table 2. Mean delta Cq values of miRNA-profiling and longitudinal analysis of miR-122, miR-210 and miR-532-5p levels in miravirsen and placebo dosed patients

Miravirsen Placebo P-value

P-value (Benjamini-Hochberg)

Patients, n 12 4 N.A. N.A.

miR-122 profiling Baseline (log10 2 - ∆Cq) 0.80 ± 0.42 0.64 ± 0.65 0.58 0.85 Week 4 (log10 2 - ∆Cq) - 2.64 ± 0.48 0.74 ± 0.49 7.94E-09 1.33E-06 FC week 4 (log10) - 3.44 ± 0.53 0.09 ± 0.21 4.02E-09 7.64E-08

miR-210 profiling Baseline (log10 2 - ∆Cq) - 0.79 ± 0.25 - 0.80 ± 0.19 0.98 0.99 Week 4 (log10 2 - ∆Cq) - 1.26 ± 0.25 - 0.63 ± 0.11 0.0002 0.02 FC week 4 (log10) - 0.47 ± 0.38 0.17 ± 0.18 0.007 0.06 Longitudinal analysis FC week 1 (log10) 0.18 ± 0.66 0.99 ± 1.34 0.69 N.A. FC week 4 (log10) - 0.17 ± 0.52 1.05 ± 1.40 0.12 N.A. FC week 6 (log10) - 0.41 ± 0.72 0.34 ± 1.89 0.79 N.A. FC week 10/12 (log10) - 0.05 ± 0.73 0.46 ± 1.90 0.48 N.A.

miR-532-5p profiling Baseline (log10 2 - ∆Cq) - 1.10 ± 0.18 - 0.81 ± 0.25 0.03 0.55 Week 4 (log10 2 - ∆Cq) - 1.77 ± 0.59 - 0.73 ± 0.41 0.006 0.17 FC week 4 (log10) - 0.67 ± 0.55 0.08 ± 0.25 0.02 0.11 Longitudinal analysis FC week 1 (log10) 0.30 ± 0.84 0.01 ± 0.72 0.55 N.A. FC week 4 (log10) 0.16 ± 0.71 0.24 ± 0.42 0.84 N.A. FC week 6 (log10) - 0.89 ± 0.83 - 0.48 ± 0.83 0.42 N.A. FC week 10/12 (log10) - 0.51 ± 1.07 - 0.49 ± 1.27 0.98 N.A.

Data are given as mean values ± SD. FS, fold change. N.A., not applicable.

miravirsen during the study period, and were not significantly affected by the inhibition

of miR-122.

MiR-122 is the most abundant hepatocellular miRNA, with ~66,000 copies per cell 30,

and is required for efficient HCV replication. MiR-122 positively affects HCV RNA stability,

translation and replication, but the exact mechanism involved is not fully understood.

Interestingly, earlier studies showed that serum and hepatic miR-122 levels, and serum

miR-122 and HCV RNA levels are not associated 31,32. In addition, it is unknown how many

miR-122 copies are required for efficient HCV replication. In this study, we show that all

CHAPTER 2

42

Figure 4. MiRNA profiling results. (A) Flowchart of analysis and selection of miRNAs. (B) Hierarchal clustering of patients and a selection of miRNAs with fold change ≥ 2 or ≤ 0.5 in patients dosed with miravirsen and fold change level between 0.5 and 2 in placebo treated patients (n=19). The color scale illustrates the relative expression of a miRNA across all samples: yellow color represents an high expression, blue color represent low expression. Unsupervised clustering clustered patients dosed with miravirsen and placebo in separate groups. * P < 0.05 between study groups.

A)

B)

PLASMA MIRNA-LEVELS FOLLOWING MIRAVIRSEN DOSING

43

2

B)

A)

Figure 5. Fold change in plasma miR-210 and miR-532-5p level. Fold change in miRNA-level at week 1, 4, 6 and 10/12 compared to baseline in patients dosed with placebo or miravirsen. Longitudinal change in (A) plasma miR-210 levels and (B) plasma miR-532-5p levels in patients dosed with miravirsen compared to placebo. Figure showing mean and SEM; ns: not significant.

patients dosed with miravirsen had a significant reduction in plasma miR-122 levels, and

that miR-122 levels became undetectable in some patients. However, this was not always

accompanied by a substantial reduction in the viral load, and there was no significant

correlation between the fold change in plasma miR-122 and change in HCV RNA levels

at week 4 as compared to baseline. Recently, it was demonstrated that a C3U nucleotide

change in the 5’ UTR of the HCV RNA appeared in patients with virological relapse after

miravirsen dosing 26. The C3U viral mutant had a decreased fitness and was insensitive to

miravirsen, but fully susceptible to other anti-HCV agents. It was postulated that the C3U

mutant renders the virus independent of miR-122, suggesting that this mutant uses an

alternative mechanism to support RNA stabilisation and replication.

CHAPTER 2

44

It has previously been shown that an elevated serum miR-122 level is a sensitive marker

for inflammatory activity in the liver and that it is associated with ALT level 33,34. It was

postulated that the presence of circulating miR-122 may be the result of normal hepatocyte

turnover, and that increased miR-122 level are caused by hepatocyte lysis 35. In addition, an

inverse relation between the serum miR-122 level and the stage of fibrosis was described,

possibly due to a decreased number of functional hepatocytes in liver cirrhosis 33,35,36.

However, the presence of miR-122 in plasma could also be the result of active secretion by

the hepatocyte, either associated with RNA-binding proteins and lipoprotein complexes

or packaging into micro-particles 37–39. In this study, we demonstrate a rapid decrease in

plasma miR-122 levels in patients dosed with miravirsen. One week after the first injection,

the plasma miR-122 level was significantly decreased compared to baseline, and this

remained low throughout the study period. In our study, we did not find a significant

correlation between plasma miR-122 and ALT level at baseline. However, there was an

association between change in plasma miR-122 and ALT level 4 weeks after the first dose

of miravirsen. The lower plasma miR-122 levels after miravirsen dosing could be the result

of less hepatocyte injury, or a diminished active secretion of miR-122 by targeting

hepatic miR-122.

MiR-122 is believed to have a tumour suppressive role and has been related to

the development of HCC 16,40. In adult mice, a short-term inhibition of miR-122 using

an antisense oligonucleotide for 5 weeks was well tolerated, and these mice did not

develop HCC 18. Despite our follow-up study wherein no safety problems were observed

in miravirsen-dosed patients 25, the exact long-term risk of HCC development needs to be

elucidated. To better valuate the risk of transient miR-122 inhibition on HCC development,

we assessed how long plasma miR-122 levels remained decreased after miravirsen

dosing in CHC patients. We observed an increasing trend of plasma miR-122 levels until

approximately 2 months (week 10/12) after the last dose of miravirsen (at week 4), in

patients dosed with 3 and 5 mg/kg miravirsen. Plasma miR-122 levels of the majority of

patients dosed with 7 mg/kg miravirsen were still decreasing at week 10/12, indicating

a dose-dependent difference in the duration of miR-122 inhibition. Taken together, these

data suggest that there is a prolonged inhibitory effect on miR-122 but that miR-122 levels

are expected to return to baseline over time. The prolonged effect on miR-122 levels may

be explained by the long tissue half-life or miravirsen (~30 days), and that miravirsen does

not only target mature miR-122, but was shown to also suppress the biogenesis of miR-122

at the primary and precursor miRNA levels in vitro 41.

In this study, we assessed whether antagonising miR-122 alters the expression level of

other miRNAs that are found in plasma. MiRNAs are involved in regulating various cell

functions and homoeostasis, they function by targeting the 3’ or 5’ UTR of mRNA and

thereby degrade their target or its translation. Each miRNA can bind multiple target

mRNAs, and each mRNA has numerous binding sites for multiple miRNAs. Furthermore,

PLASMA MIRNA-LEVELS FOLLOWING MIRAVIRSEN DOSING

45

2

miRNAs are known to have a tremendous interplay with each other and expression is

tightly controlled by both transcriptional and post-transcriptional regulatory systems. Next

to those for miR-122, binding sites for several miRNAs have been identified in the HCV

RNA genome 10,42,43. In contrast to the stimulatory effect of miR-122 on HCV, miR-199a,

let-7b, miR-196b and miR-491 were shown to inhibit HCV replication in vitro 44–48. We

showed that antagonising miR-122 solely results in reduced plasma miR-122 levels and that

plasma levels of other miRNAs are not significantly affected. Furthermore, we observed

substantial variations in plasma miRNA levels during the study period in both placebo and

miravirsen dosed patients. Unfortunately, little is known about normal miRNA levels and

their variability in humans, and especially in CHC patients. Since miRNAs are important

players in regulating various cellular processes, the unchanged miRNA-profile (except

miRNA-122) indicates that miravirsen targets exclusively miRNA-122, which is favourable

from the perspective of safety.

A limitation of this study is the small number of patients which is due to the fact that

additional blood samples were only collected in a selection of patients dosed with

miravirsen. Since no liver biopsies were taken prior and after miravirsen dosing, we can only

speculate about changes in hepatic miRNA levels in these patients. However, as observed

with the substantial reduction in plasma miR-122 levels, we believe that important changes

in tissue-specific miRNA levels will be reflected in the plasma. Nevertheless, this study

provides important information on changes in plasma miRNA levels in the first cohort of

patients dosed with an anti-miRNA oligonucleotide.

We have used the mean expression values of miR-93 and miR-191 for normalisation of

plasma miR-122 levels. However, miR-191 has previously been described to be slightly

elevated in serum of patients with CHC compared to that of healthy controls 33. Currently,

there is no consensus which miRNA to use to establish normalisation of plasma miRNA

levels. Nevertheless, we believe that our reference miRNAs are justified in this study since

miR-191 was stable in our cohort and that absolute (without normalisation) and relative

(with normalisation) miR-122 levels showed the same pattern.

The therapeutic field in hepatitis C treatment is changing quickly with the registration and

ongoing development of several DAA’s. Miravirsen is the first anti-miRNA oligonucleotide

that has been administered in humans, and in particular in CHC patients. Although

the development of miravirsen has ceased, a Gal-NAc conjugated anti-miR-122

oligonucleotide with a 20-fold higher potency compared to miravirsen is currently being

investigated in clinical trials 49. This study evaluated changes in plasma miRNA levels after

anti-miR-122 oligonucleotide dosing. We demonstrated that administration of various

doses of miravirsen resulted in a substantial and prolonged decrease in plasma miR-122

levels and did not affect plasma levels of other miRNAs in CHC patients.

CHAPTER 2

46

REFERENCES1. Ambros V. The functions of animal microRNAs.

Nature. 2004 Sep 16;431(7006):350–5.

2. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004 Jan 23;116(2):281–97.

3. Shukla GC, Singh J, Barik S. MicroRNAs: Processing, Maturation, Target Recognition and Regulatory Functions. Mol Cell Pharmacol. 2011 Jan;3(3):83–92.

4. Bushati N, Cohen SM. microRNA functions. Annu Rev Cell Dev Biol. 2007 Jan;23:175–205.

5. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993 Dec 3;75(5):843–54.

6. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell. 1993 Dec 3;75(5):855–62.

7. Reid G, Kirschner MB, van Zandwijk N. Circulating microRNAs: Association with disease and potential use as biomarkers. Crit Rev Oncol Hematol. 2011 Nov;80(2):193–208.

8. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 2008;105(30):10513–8.

9. Lindenbach BD, Rice CM. Unravelling hepatitis C virus replication from genome to function. Nature. 2005 Aug 18;436(7053):933–8.

10. van der Ree MH, de Bruijne J, Kootstra NA, Jansen PL, Reesink HW. MicroRNAs: role and therapeutic targets in viral hepatitis. Antivir Ther. 2014 Jan;19(6):533–41.

11. Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science. 2005 Sep 2;309(5740):1577–81.

12. 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 Jan 8;327(5962):198–201.

13. Sedano CD, Sarnow P. Hepatitis C Virus Subverts Liver-Specific miR-122 to Protect the Viral Genome from Exoribonuclease Xrn2. Cell Host Microbe. 2014 Aug 13;16(2):257–64.

14. Li Y, Yamane D, Lemon SM. Dissecting the roles of the 5’ exoribonucleases Xrn1 and Xrn2 in restricting hepatitis C virus replication. J Virol. 2015 May 11;89(9):4857–65.

15. Machlin ES, Sarnow P, Sagan SM. Masking the 5’ terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex. Proc Natl Acad Sci U S A. 2011 Feb 22;108(8):3193–8.

16. Coulouarn C, Factor VM, Andersen JB, Durkin ME, Thorgeirsson SS. Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. Oncogene. 2009 Oct 8;28(40):3526–36.

17. Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, et al. Silencing of microRNAs in vivo with “antagomirs”. Nature. 2005 Dec 1;438(7068):685–9.

18. Esau C, Davis S, Murray SF, Yu XX, Pandey SK, Pear M, et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab. 2006 Feb;3(2):87–98.

PLASMA MIRNA-LEVELS FOLLOWING MIRAVIRSEN DOSING

47

2

19. Hajarizadeh B, Grebely J, Dore GJ. Epidemiology and natural history of HCV infection. Nat Rev Gastroenterol Hepatol. 2013 Sep 2;10(9):553–62.

20. Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med. 2001 Jul 5;345(1):41–52.

21. Backus LI, Boothroyd DB, Phillips BR, Belperio P, Halloran J, Mole L a. A sustained virologic response reduces risk of all-cause mortality in patients with hepatitis C. Clin Gastroenterol Hepatol. 2011 Jun;9(6):509–516.e1.

22. Cardoso A-C, Moucari R, Figueiredo-Mendes C, Ripault M-P, Giuily N, Castelnau C, et al. Impact of peginterferon and ribavirin therapy on hepatocellular carcinoma: incidence and survival in hepatitis C patients with advanced fibrosis. J Hepatol. 2010 May;52(5):652–7.

23. van der Meer A, Veldt BJ, Feld JJ, Wedemeyer H, Dufour J-F, Lammert F, et al. Association between sustained virological response and all-cause mortality among patients with chronic hepatitis C and advanced hepatic fibrosis. JAMA. 2012 Dec 26;308(24):2584–93.

24. Janssen HLA, Reesink HW, Lawitz EJ, Zeuzem S, Rodriguez-Torres M, Patel K, et al. Treatment of HCV infection by targeting microRNA. N Engl J Med. 2013 May 2;368(18):1685–94.

25. van der Ree MH, van der Meer AJ, de Bruijne J, Maan R, Van Vliet A, Welzel TM, et al. Long-term safety and efficacy of microRNA-targeted therapy in chronic hepatitis C patients. Antiviral Res. 2014;111:53–9.

26. Ottosen S, Parsley TB, Yang L, Zeh K, van Doorn L-J, van der Veer E, et al. In vitro antiviral activity and preclinical and clinical resistance profile of miravirsen, a novel anti-hepatitis C virus therapeutic targeting the human factor

miR-122. Antimicrob Agents Chemother. 2015 Jan 1;59(1):599–608.

27. Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004;64:5245–50.

28. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:RESEARCH0034.

29. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008 Jan;3(6):1101–8.

30. Chang J, Nicolas E, Marks D, Sander C, Lerro A, Buendia MA, et al. miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1. RNA Biol. 2004 Jul;1(2):106–13.

31. Morita K, Taketomi A, Shirabe K, Umeda K, Kayashima H, Ninomiya M, et al. Clinical significance and potential of hepatic microRNA-122 expression in hepatitis C. Liver Int. 2011 Apr;31(4):474–84.

32. Cermelli S, Ruggieri A, Marrero JA, Ioannou GN, Beretta L. Circulating microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver disease. PLoS One. 2011 Jan;6(8):e23937.

33. van der Meer AJ, Farid WRR, Sonneveld MJ, de Ruiter PE, Boonstra A, van Vuuren AJ, et al. Sensitive detection of hepatocellular injury in chronic hepatitis C patients with circulating hepatocyte-derived microRNA-122. J Viral Hepat. 2013 Mar 27;20(3):158–66.

CHAPTER 2

48

34. Bihrer V, Friedrich-Rust M, Kronenberger B, Forestier N, Haupenthal J, Shi Y, et al. Serum miR-122 as a biomarker of necroinflammation in patients with chronic hepatitis C virus infection. Am J Gastroenterol. 2011 Sep;106(9):1663–9.

35. Trebicka J, Anadol E, Elfimova N, Strack I, Roggendorf M, Viazov S, et al. Hepatic and serum levels of miR-122 after chronic HCV-induced fibrosis. J Hepatol. 2013 Feb;58(2):234–9.

36. Marquez RT, Bandyopadhyay S, Wendlandt EB, Keck K, Hoffer BA, Icardi MS, et al. Correlation between microRNA expression levels and clinical parameters associated with chronic hepatitis C viral infection in humans. Lab Invest. 2010 Dec;90(12):1727–36.

37. Creemers EE, Tijsen AJ, Pinto YM. Circulating MicroRNAs: Novel biomarkers and extracellular communicators in cardiovascular disease? Vol. 110, Circulation Research. 2012. p. 483–95.

38. Roderburg C, Luedde T. Circulating microRNAs as markers of liver inflammation, fibrosis and cancer. J Hepatol. 2014 Dec;61(6):1434–7.

39. Cortez MA, Bueso-Ramos C, Ferdin J, Lopez-Berestein G, Sood AK, Calin GA. MicroRNAs in body fluids--the mix of hormones and biomarkers. Nat Rev Clin Oncol. 2011;8:467–77.

40. Bai S, Nasser MW, Wang B, Hsu S-H, Datta J, Kutay H, et al. MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to sorafenib. J Biol Chem. 2009 Nov 13;284(46):32015–27.

41. Gebert LFR, Rebhan M a E, Crivelli SEM, Denzler R, Stoffel M, Hall J. Miravirsen (SPC3649) can inhibit the biogenesis of miR-122. Nucleic Acids Res. 2014 Jan 24;42(1):609–21.

42. Conrad KD, Niepmann M. The role of microRNAs in hepatitis C virus RNA replication. Arch Virol. 2013 Oct 25;

43. Sedano CD, Sarnow P. Interaction of host cell microRNAs with the HCV RNA genome during infection of liver cells. Semin Liver Dis. 2015 Feb;35(1):75–80.

44. Murakami Y, Aly HH, Tajima A, Inoue I, Shimotohno K. Regulation of the hepatitis C virus genome replication by miR-199a. J Hepatol. 2009 Mar;50(3):453–60.

45. Cheng J-C, Yeh Y-J, Tseng C-P, Hsu S-D, Chang Y-L, Sakamoto N, et al. Let-7b is a novel regulator of hepatitis C virus replication. Cell Mol Life Sci. 2012 Aug;69(15):2621–33.

46. Pedersen IM, Cheng G, Wieland S, Volinia S, Croce CM, Chisari F V, et al. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature. 2007 Oct 18;449(7164):919–22.

47. Liu Z, Tian Y, Machida K, Lai MMC, Luo G, Foung SKH, et al. Transient activation of the PI3K-AKT pathway by hepatitis C virus to enhance viral entry. J Biol Chem. 2012 Dec 7;287(50):41922–30.

48. Ishida H, Tatsumi T, Hosui A, Nawa T, Kodama T, Shimizu S, et al. Alterations in microRNA expression profile in HCV-infected hepatoma cells: involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway. Biochem Biophys Res Commun. 2011 Aug 19;412(1):92–7.

49. Van Der Ree M, de Vree ML, Stelma F, Willemse S, van der Valk M, Rietdijk S, et al. LO7: A single subcutaneous dose of 2mg/kg or 4mg/kg of RG-101, a GalNAc-conjugated oligonucleotide with antagonist activity against MIR-122, results in significant viral load reductions in chronic hepatitis C patients. J Hepatol. 2015 Apr 4;62:S261.

PLASMA MIRNA-LEVELS FOLLOWING MIRAVIRSEN DOSING

49

2

ACKNOWLEDGEMENTS We thank professor C.P. Engelfriet for critical review of this manuscript.

FINANCIAL SUPPORTThis investigator initiated study was partly funded by Santaris Pharma A/S,

Hørsholm, Denmark.