SQU Med J, December 2010, Vol. 10, Iss. 3, pp. 312-317, Epub. 14th Nov 10 
Invited Article
Submitted 17th April 10
Revision ReQ. 20th June 10, Revision recd. 6th July 10
Accepted 13th July 10

The hepatitis C virus (HCV) is a worldwide public health problem affecting an estimated 200 million individuals. There 
are approximately 3–4 million new cases of HCV 
infection each year. HCV is a serious infectious 
problem in the Middle East with Egypt having the 
highest prevalence of HCV infection and Oman 
being in the intermediate category.1,2

HCV is a flavivirus comprising 6 genotypes 
with numerous subtypes. Genotype 1 is the most 
prevalent in Europe and North America and the 
most difficult to treat. Genotypes 2 and 3 appear 
to be more prevalent in the Far East. Of the other 
genotypes, genotype 4 is common in Africa and 
the Middle East, whereas genotypes 5 and 6 are 
predominant in South Africa and South-East Asia, 
respectively.3-5 

Only 10% - 40% of acute HCV-infected 
individuals can be cured by spontaneous viral 

clearance and 60% - 90% of infected individuals 
develop chronic infection.6 There is a very slight 
chance of clearing the virus spontaneously in 
chronic HCV carriers (0.5% - 0.74%); however, 
the majority of patients with chronic hepatitis 
C infection will not clear the infection without 
treatment. Progression of liver diseases usually takes 
decades and up to 20% of those infected may develop 
complications including cirrhosis, liver failure, or 
hepatocellular carcinoma.7  End-stage liver disease, 
due to infection with HCV, currently represents the 
major indication for liver transplantation, and the 
virus universally recurs after transplantation. 

Acute HCV infection is characterised by a delay 
of 6–8 weeks in the induction of adaptive immunity. 
This is despite active viral replication; this therefore 
suggests the failure of innate immunity to contain 
viral replication and the presence of a blockade 
in the cross-talk between innate and adaptive 

Department of Microbiology & Immunology, Faculty of Medicine, Montreal University; Montreal, Quebec, Canada; and Montreal 
University Hospital Research Center (CR-CHUM), St-Luc Hospital, Montreal, Quebec, Canada. 
Email: elias_a_said@yahoo.fr

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aBstract: Infection with the hepatitis C virus (HCV) is a worldwide problem. Patients with chronic HCV 
infection who are non-responders to standard therapy represent a growing population within the HCV epidemic. 
Novel, more efficient and tolerable therapies are urgently needed. This review discusses the recent results showing 
that targeting miR-122, a micro-ribonucleic acid (MicroRNA) that enhances HCV replication, is a new anti-HCV 
therapy with a high barrier to resistance.

Keywords: Hepatitis C virus; MicroRNAs; Interferon; Liver; Infection; Therapy.

REVIEW

The Need for New Anti-Hepatitis C Virus 
Therapeutic Strategies

Targeting the cellular micro-ribonucleic acids?
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review | 313

immunity. CD8+ (cluster of differentiation 8) 
and CD4+ (cluster of differentiation 4) HCV-
specific T-cell responses are essential to prevent 
viral persistence8,9 and it has been demonstrated 
that infections that progress to chronicity are 
characterised by a limited immune response either 
in frequency, magnitude or the number of effector 
functions that can be detected.10 Unfortunately, 
the mechanisms underlying the inefficient priming 
of HCV-specific T-cells in infections with chronic 
evolution are poorly understood. 

In this review we will discuss how targeting 
MicroRNA-122 (miR-122), which enhances HCV 
replication with specific oligonucleotidic sequences, 
can be a new anti-HCV therapy with a high barrier 
to resistance.

The Efficacy of Current 
Anti-HCV Treatments
There is currently no effective anti-HCV vaccine 
available. The use of interferon-α (IFN-α) for 
treatment was based on the fact that this cytokine 
is an important element of the innate antiviral 
immune response; however, success with the IFN-
based therapy was modest. Treatment with IFN-α 
induced a quick decrease of HCV ribonucleic 
acid (RNA) levels in the serum, and the long-
term responses were characterised by a sustained 
absence of HCV RNA from the serum and liver, 
and resolution of the chronic infection. The use of 
ribavirin and pegylated (peg-) IFN-α, where IFN-α 
was linked to polyethylene glycol (peg) in order to 
increase the half-life of IFN, significantly increased 
the proportion of patients achieving sustained 
antiviral response (SVR).6,7 Long-term studies have 
shown that SVR indicates clearance of the virus and 
cure of the disease. Unfortunately, although anti-
HCV therapy combining peg-IFN-α with ribavirin 
has significantly improved, it leads to sustained 
clearance in only about 50% of patients and the 
treatment is frequently associated with severe 
side effects.6,7 Multiple factors can modulate the 
response to therapy, i.e. viral genotype and patient 
characteristics. Rates of SVR range from 42% - 46% 
in patients with genotype 1. In contrast, SVR rates 
for patients with genotypes 2 and 3 are 76 - 80% and 
the clearance can be reached with a shorter course 
of therapy and lower doses of ribavirin. The host 
determinants of therapy are still not well known. 

Certain patient populations hardly respond to 
treatment. A transient response with relapse occurs 
in 10 - 25% of patients. Non-response to treatment 
occurs in about one-third of chronic HCV-infected 
patients. These patients never reach clearance, 
although viral loads might fall at some stage in 
treatment.6,7 However, treatment in the acute 
phase of the infection is accompanied by a high 
rate of response where IFN-α therapy reduces the 
chronicity rate to 10% or lower. Nonetheless, non-
responders among the HCV-infected represent a 
growing population within the HCV epidemic. The 
majority of these patients is frequently labelled as 
“difficult to treat”. They are infected with genotype 
1 and have a high viral load and advanced fibrosis 
or cirrhosis. The best therapy available has been 
unsuccessful in inducing a virologic response in 
these patients. Clarification of the reasons of non-
response to IFN-α therapy is a main challenge to 
research on HCV. 

The mechanism of how IFN-α inhibits HCV 
replication remains poorly understood. It has been 
reported that IFN-α has potent antiviral activity, but 
does not act directly on the virus or the replication 
complex. Instead, it acts by inducing IFN-stimulated 
genes (ISGs), which establish a non-virus-specific 
antiviral state within the cell.7 Recently, microarray 
analyses were performed on liver biopsies taken 
before therapy from a cohort of HCV-infected 
patients treated with peg-IFN-α and ribavirin. The 
results showed that patients who were subsequently 
identified as non-responders had higher baseline 
expression of ISGs than responders to therapy (and 
those who relapsed); the latter resembled healthy 
controls more closely. Interestingly, peg-IFN-α did 
not induce the expression of ISGs to levels higher 
than those observed at the pre-treatment stage 
in the non-responders.7 These findings suggest 
that non-responders have an up-regulated and 
largely ineffective IFN response. Coincidentally, 
chimpanzees with chronic HCV infection have high 
expression levels of ISGs; however, neither type-1 
nor type-2 IFNs are induced during the infection.11,12 
Although IFN-α could induce ISGs expression in 
healthy chimpanzees, those chronically infected 
do not respond to IFN treatment and thus 
appear to be an extreme representative of non-
responders in human HCV-infected patients.  
This makes chimpanzees an ideal model for the 
study of non-responsiveness to IFN-α therapy and 



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314 | SQU Medical Journal, December 2010, Volume 10, Issue 3

therapy (cART).17-19 Therapies that target essential 
host functions for HCV may provide a high barrier 
to resistance and, thus, could present an effective 
approach for the development of new HCV antiviral 
drugs.

Sequestrating Cellular 
MicroRNAs is a Promising 
Strategy
New hope can be given by targeting cellular 
microRNAs that are implicated in the HCV life 
cycle. MicroRNAs are a class of small non-coding 
RNA molecules that function through post-
transcriptional regulation of gene expression. 
Those small RNAs are important in development 
and disease.20 Therefore, they represent a potential 
new class of targets for therapeutic intervention. 
MicroRNA 122 (MiR-122) is specifically expressed 
in the liver, where it constitutes 70% of the total 
microRNA population.21 Three miR-122 binding 
sites in the HCV genome have been reported: one 
is located in 3’ non-coding region (NCR) of the 
HCV genome and its function remains unknown; 
the other two binding sites are closely located in 
the 5’ NCR and separated by a highly conserved 
14-nucleotides sequence.21 Those sequences are 
highly conserved among the six HCV genotypes. 
MiR-122 binds to sequences in the 5’ NCR of HCV 
RNA, i.e. S1 and S2, resulting in the up-regulation 
of viral RNA levels [Figure 1].22 Interaction of miR-
122 with the HCV genome is essential for the 
accumulation of viral RNA in the cultured liver cells 
and all target sites are required for modulation of 
HCV RNA abundance. Although treatment with 
IFN has been shown to decrease miR-122 levels, no 
correlation between miR-122 levels in the liver and 
the viral load was observed upon treatment with 
IFN.23,24 However, lower levels of miR-122 were 
observed in non-responders.24 Of note, the CD56+ 
T-cells were shown to inhibit HCV replication by 
decreasing miR-122 expression. Whether this effect 
is due to the up-regulation of type-I IFN expression 
by hepatocytes remains to be clarified.25

RNA oligonucleotides with exact 
complementarity to miR-122 have been shown 
to sequester miR-122 leading to significant 
decrease in HCV RNA accumulation [Figure 1].  
This observation underscored miR-122 as a  
potential therapeutic target for the treatment of  

for the development of novel antiviral agents.
Overall, although IFN therapy has considerably 

improved, 50% of the infected individuals with 
chronic disease do not achieve sustained clearance 
of HCV. Recent studies suggest that the duration of 
the treatment with peg-IFN plus ribavirin therapy 
must be determined in each patient according to the 
on-treatment virological response, i.e. the viral load 
at 4 weeks and the genotype.13 Currently, there are 
no approved treatment options available for patients 
who have failed to respond to previous treatments. 
Novel, more efficient and tolerable therapies are 
urgently needed. A greater understanding of the 
viral life cycle has led to an increase in the number 
of possible targets for antiviral intervention. It has 
resulted in the development of several agents that 
target specific stages of the life cycle. Potential 
processes for viral inhibition include virus entry 
into the host cell, proteolytic processing, RNA 
replication and the assembly and release of the 
new viral particles. Those agents include protease 
inhibitors, i.e., BILN 2061 (the trials were stopped 
due to severe side effects in the animal model); 
proceprevi SCH 503034 and telaprevir (both 
tested in phase II); polymerase inhibitors (i.e., 
valopicitabine, BILB 1941– the trials were stopped 
due to gastrointestinal-related side effects); immune 
modulators (i.e., ANA 975 for TLR7) and host 
factor inhibitors (i.e., NIM-811 for cyclophilin B). A 
very recent study showed the potential clinical anti-
HCV effect of BMS-790052, an inhibitor of NS5A, 
which is an HCV protein with no known enzymatic 
function.14 A list of other trials, including two other 
forms of IFN (albuferon [albumin IFN]) and locteron 
(a newly developed controlled-release formulation 
of lemna-derived free (unpegylated) recombinant 
interferon-α2b), can be found on the website of 
the HIV and Hepatitis Treatment Advocates, Inc.15 
However, the generation and selection of resistant 
variants can allow the virus to escape the antiviral 
pressure exerted by treatment. Indeed, mutations 
in both the polymerase and protease enzymes have 
already been identified.16 The HIV infection model 
suggests that the combination of different anti-viral 
therapies might decrease HCV chances to develop 
escape mutations; however recent studies on HIV-
infected patients demonstrated the emergence of 
drug resistance during receipt of combined therapy 
and described a relatively common virological 
failure by 8 years of combination antiretroviral 



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review | 315

HCV infection. Locked nucleic acid (LNA) 
technology20 has been introduced into the 
development of an efficient approach for miR-
122 targeting in vivo and high-affinity LNA-
modified DNA oligonucleotides (LNA anti-miRs) 
complementary to miR-122 were developed.26 
LNA-modified nucleosides are a class of nucleic 
acid analogues in which the ribose ring is 
“locked” by a methylene bridge connecting the 
2’-O atom and the 4’-C atom. By “locking” the 
molecule with the methylene bridge, the LNA-
modified nucleosides are constrained in the ideal 
conformation for Watson-Crick binding.20 When 
incorporated into a DNA oligonucleotide, LNA 
therefore enhances the speed of the pairing with a 
complementary nucleotide strand and increases the 
stability of the resulting duplex. Phosphorothioate 
modifications have been shown to provide good 
pharmacokinetic and tissue uptake properties in 
antisense oligonucleotides along with protection 
against nucleases. Moreover, it has been reported 
that specific miR-122 silencing in vivo using a 

LNA-modified phosphorothioate oligonucleotide 
(SPC3649) complementary to the 5’-end of miR-122 
leads to long-lasting decrease of serum cholesterol 
in mice and African green monkeys, which make 
it possible to inhibit the miR-122 function in vivo. 
Thus, it was tempting to investigate the potential of 
miR-122 antagonism as a new anti-HCV therapy.

The potential role of SPC3649 as a new anti-
HCV treatment was tested in chronically HCV-
infected chimpanzees.26 Treatment with high doses 
of SPC3649 induced a decrease of 2.3 log10 in HCV 
RNA levels in the liver of the animals. These results 
showed that MiR-122 is essential for accumulation 
of HCV RNA in vivo. Moreover, the absence of 
viral resistance to therapy during treatment with 
SPC3649 has been demonstrated. This lack of viral 
resistance to SPC3649 is in striking contrast to what 
has been observed with direct acting antiviral drugs 
in HCV-infected chimpanzees. Within two days 
of treatment with a non-nucleoside polymerase 
inhibitor, 67% of the HCV clones already possessed 
known resistance mutations, with 10% of the clones 

Figure 1: The binding of MiR-122 to hepatitis C virus (HCV) genome and its inhibition by SPC3649. The attachment 
of miR-122 to target sites, S1 and S2, in the 5’UTR of HCV ribonucleic acid (RNA) induces an increase in the amount 
of viral RNA and thus viral replication. The locked nucleic acid (LNA)-anti-miR-122, SPC3649, binds to MiR-122 and 
prevents its interaction with HCV RNA, which inhibits the replication of HCV.



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316 | SQU Medical Journal, December 2010, Volume 10, Issue 3

currently being explored and could decrease HCV 
replication.27 Of note, two other microRNAs, 
miR199a and miR-196, were identified as possible 
targets for anti-HCV therapy as, in contrast to miR-
122, the overexpression of these two molecules 
leads to the inhibition of HCV replication in the 
replicon system in vitro.28,29 However, the effect of 
these molecules should be validated on real HCV 
infection in vitro prior to testing them in the animal 
model and then in clinical trials.

Conclusion
Many therapeutic strategies against HCV infection 
are available, such as the combination of peg-IFN 
and ribavirin. However, the failure of these therapies, 
because of viral escape or the non-response of 
many patients to these therapies, makes the use of 
LNA-modified phosphorothioate oligonucleotide 
(SPC3649), which blocks miR-122, a very potent 
anti-HCV treatment with no side effects or escape 
mutations. 

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