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