• COVID-19
• SARS-CoV-2
• coronaviruses
• dsRNA
• nucleic acid therapy
• patents
• siRNA delivery
• siRNA therapy

• Antiviral Agents/history
• Antiviral Agents/therapeutic use
• COVID-19/epidemiology
• COVID-19/history
• COVID-19/virology
• Clinical Trials as Topic
• Drug Approval
• Drug Evaluation, Preclinical
• History, 20th Century
• History, 21st Century
• Humans
• Mutation
• Pandemics/prevention & control
• Patents as Topic
• RNA, Small Interfering/history
• RNA, Small Interfering/therapeutic use
• SARS-CoV-2/genetics
• SARS-CoV-2/pathogenicity
• COVID-19 Drug Treatment

• School of Pharmacy, Chapman University

Being the most conserved region of all hepatitis C virus (HCV) genotypes and sub-genotypes, the 5′ untranslated region (5′ UTR) of HCV genome signifies it’s importance as a potential target for anti-mRNA based treatment strategies like RNA interference. The advent and approval of first small interference RNA (siRNA) -based treatment of hereditary transthyretin-mediated amyloidosis for clinical use has raised the hopes to test this approach against highly susceptible viruses like HCV. We investigated the antiviral potential of consensus siRNAs targeted to stem-loops (SLs) II and III nucleotide motifs of internal ribosome entry site (IRES) structure within 5′ UTR of HCV sub-genotype 4a isolates from the Saudi population. siRNA inhibitory effects on viral replication and translation of full-length HCV genome were determined in a competent, persistent, and reproducible Huh-7 cell culture system maintained for one month. Maximal inhibition of RNA transcript levels of HCV-IRES clones and silencing of viral replication and translation of full-length virus genome was demonstrated by siRNAs targeted to SL-III nucleotide motifs of IRES in Huh-7 cells. siRNA Usi-169 decreased 5′ UTR RNA transcript levels of HCV-IRES clones up to 75% (P < 0.001) at 24 h post-transfection and 80% (P < 0.001) at 48 h treatment in Huh-7 cells. 5′ UTR-tagged GFP protein expression was significantly decreased from 70 to 80% in Huh-7 cells co-transfected with constructed vectors (i.e. pCR3.1/GFP/5′ UTR) and siRNA Usi-169 at 24 h and 48 h time-span. Viral replication was inhibited by more than 90% (P < 0.001) and HCV core (C) and hypervariable envelope glycoproteins (E1 and E2) expression was also significantly degraded by intracytoplasmic siRNA Usi-169 activity in persistent Huh-7 cell culture system. The findings unveil that siRNAs targeted to 5′ UTR-IRES of HCV sub-genotype 4a Saudi isolates show potent silencing of HCV replication and blocking of viral translation in a persistent in-vitro Huh-7 tissue culture system. Furthermore, we also elucidated that siRNA silencing of viral mRNA not only inhibits viral replication but also blocks viral translation. The results suggest that siRNA potent antiviral activity should be considered as an effective anti-mRNA based treatment strategies for further in-vivo investigations against less studied and harder-to-treat HCV sub-genotype 4a isolates in Saudi Arabia.

• 5′ Untranslated region
• Anti-mRNA based treatment strategies
• HCV
• HCV therapeutics
• Internal ribosome entry sites
• Replication
• Small interference RNA
• Translation

• Hepatitis C virus
• RNA interference
• non-structural 5B protein
• real-time polymerase chain reaction
• stable human hepatoma-7 cells

Viral diseases like influenza, AIDS, hepatitis, and Ebola cause severe epidemics worldwide. Along with their resistant strains, new pathogenic viruses continue to be discovered so creating an ongoing need for new antiviral treatments. RNA interference is a cellular gene‐silencing phenomenon in which sequence‐specific degradation of target mRNA is achieved by means of complementary short interfering RNA (siRNA) molecules. Short interfering RNA technology affords a potential tractable strategy to combat viral pathogenesis because siRNAs are specific, easy to design, and can be directed against multiple strains of a virus by targeting their conserved gene regions. In this review, we briefly summarize the current status of siRNA therapy for representative examples from different virus families. In addition, other aspects like their design, delivery, medical significance, bioinformatics resources, and limitations are also discussed.

• RNAi
• antiviral
• clinical trials
• delivery
• design
• limitations
• resources
• siRNA
• virus

• HCV therapeutics
• Hepatitis C virus
• NS3 protein
• NS5B
• RNA interference
• Stable Huh-7 cell culture system

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that mediates the sequence-specific degradation of targeted RNA and thus provides a tremendous opportunity for development of oligonucleotide-based drugs. Here, we report on the design and validation of small interfering RNAs (siRNAs) targeting highly conserved regions of the hepatitis C virus (HCV) genome. To aim for therapeutic applications by optimizing the RNAi efficacy and reducing potential side effects, we considered different factors such as target RNA variations, thermodynamics and accessibility of the siRNA and target RNA, and off-target effects. This aim was achieved using an in silico design and selection protocol complemented by an automated MysiRNA-Designer pipeline. The protocol included the design and filtration of siRNAs targeting highly conserved and accessible regions within the HCV internal ribosome entry site, and adjacent core sequences of the viral genome with high-ranking efficacy scores. Off-target analysis excluded siRNAs with potential binding to human mRNAs. Under this strict selection process, two siRNAs (HCV353 and HCV258) were selected based on their predicted high specificity and potency. These siRNAs were tested for antiviral efficacy in HCV genotype 1 and 2 replicon cell lines. Both in silico-designed siRNAs efficiently inhibited HCV RNA replication, even at low concentrations and for short exposure times (24h); they also exceeded the antiviral potencies of reference siRNAs targeting HCV. Furthermore, HCV353 and HCV258 siRNAs also inhibited replication of patient-derived HCV genotype 4 isolates in infected Huh-7 cells. Prolonged treatment of HCV replicon cells with HCV353 did not result in the appearance of escape mutant viruses. Taken together, these results reveal the accuracy and strength of our integrated siRNA design and selection protocols. These protocols could be used to design highly potent and specific RNAi-based therapeutic oligonucleotide interventions.

• Antiviral Agents/pharmacology
• Binding Sites
• Cell Line, Tumor
• Computer Simulation
• Conserved Sequence/genetics
• Genome, Viral
• Genotype
• Green Fluorescent Proteins/metabolism
• Hepacivirus/drug effects
• Hepacivirus/genetics
• Humans
• Internal Ribosome Entry Sites/genetics
• Luciferases/metabolism
• Nucleic Acid Conformation
• RNA, Small Interfering/therapeutic use
• RNAi Therapeutics
• Recombinant Fusion Proteins/metabolism
• Replicon/genetics
• Reproducibility of Results
• Time Factors
• Viral Nonstructural Proteins
• Virus Replication/drug effects

• National Research Foundation of Korea (KR)
• Ministry of Scientific Research and Technology, Egypt

• DNA polymerase
• Processivity factor
• Pseudorabies virus
• RNA interference
• UL42

TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034–encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5′ RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).

In this review, we discuss recent advances in nucleic acid-based therapeutic technologies that target hepatitis C virus (HCV) infection. Because the HCV genome is present exclusively in RNA form during replication, various nucleic acid-based therapeutic approaches targeting the HCV genome, such as ribozymes, aptamers, siRNAs, and antisense oligonucleotides, have been suggested as potential tools against HCV. Nucleic acids are potentially immunogenic and typically require a delivery tool to be utilized as therapeutics. These limitations have hampered the clinical development of nucleic acid-based therapeutics. However, despite these limitations, nucleic acid-based therapeutics has clinical value due to their great specificity, easy and large-scale synthesis with chemical methods, and pharmaceutical flexibility. Moreover, nucleic acid therapeutics are expected to broaden the range of targetable molecules essential for the HCV replication cycle, and therefore they may prove to be more effective than existing therapeutics, such as interferon-α and ribavirin combination therapy. This review focuses on the current status and future prospects of ribozymes, aptamers, siRNAs, and antisense oligonucleotides as therapeutic reagents against HCV.

• Hepatitis C virus
• Nucleic acid-based therapeutics
• Ribozyme
• Aptamer
• siRNA
• Antisense oligonucleotide

• Animals
• Antiviral Agents/chemistry
• Antiviral Agents/therapeutic use
• Aptamers, Nucleotide/therapeutic use
• Drug Design
• Genetic Therapy/methods
• Hepacivirus/drug effects
• Hepacivirus/genetics
• Hepacivirus/pathogenicity
• Hepatitis C/diagnosis
• Hepatitis C/genetics
• Hepatitis C/therapy
• Humans
• Nucleic Acids/chemistry
• Nucleic Acids/therapeutic use
• Oligonucleotides, Antisense/therapeutic use
• RNA Interference
• RNA, Catalytic/therapeutic use
• RNA, Small Interfering/metabolism
• Treatment Outcome

We previously identified short synthetic shRNAs (sshRNAs) that target a conserved hepatitis C virus (HCV) sequence within the internal ribosome entry site (IRES) of HCV and potently inhibit HCV IRES-linked gene expression. To assess in vivo liver delivery and activity, the HCV-directed sshRNA SG220 was formulated into lipid nanoparticles (LNP) and injected i.v. into mice whose livers supported stable HCV IRES-luciferase expression from a liver-specific promoter. After a single injection, RNase protection assays for the sshRNA and ³H labeling of a lipid component of the nanoparticles showed efficient liver uptake of both components and long-lasting survival of a significant fraction of the sshRNA in the liver. In vivo imaging showed a dose-dependent inhibition of luciferase expression (>90% 1 day after injection of 2.5 mg/kg sshRNA) with t_1/2 for recovery of about 3 weeks. These results demonstrate the ability of moderate levels of i.v.-injected, LNP-formulated sshRNAs to be taken up by liver hepatocytes at a level sufficient to substantially suppress gene expression. Suppression is rapid and durable, suggesting that sshRNAs may have promise as therapeutic agents for liver indications.

Hepatitis C Virus (HCV) and other plus-strand RNA viruses typically require the generation of a small number of negative genomes (20–100× lower than the positive genomes) for replication, making the less-abundant antigenome an attractive target for RNA interference(RNAi)-based therapy. Because of the complementarity of duplex short hairpin RNA/small interfering RNA (shRNA/siRNAs) with both genomic and anti-genomic viral RNA strands, and the potential of both shRNA strands to become part of the targeting complexes, preclinical RNAi studies cannot distinguish which viral strand is actually targeted in infected cells. Here, we addressed the question whether the negative HCV genome was bioaccessible to RNAi. We first screened for the most active shRNA molecules against the most conserved regions in the HCV genome, which were then used to generate asymmetric anti-HCV shRNAs that produce biologically active RNAi specifically directed against the genomic or antigenomic HCV sequences. Using this simple but powerful and effective method to screen for shRNA strand selectivity, we demonstrate that the antigenomic strand of HCV is not a viable RNAi target during HCV replication. These findings provide new insights into HCV biology and have important implications for the design of more effective and safer antiviral RNAi strategies seeking to target HCV and other viruses with similar replicative strategies.

The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a “single-shot ” therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.

Small hairpin RNAs (shRNAs) are widely used as gene silencing tools and typically consist of a duplex stem of 19–29 bp, a loop, and often a dinucleotide overhang at the 3′ end. Like siRNAs, shRNAs show promise as potential therapeutic agents due to their high level of specificity and potency, although effective delivery to target tissues remains a challenge. Algorithms used to predict siRNA performance are frequently used to design shRNAs as well. However, the differences between these two kinds of RNAi mediators indicate that the factors affecting target gene silencing will not be the same for siRNAs and shRNAs. Stem and loop lengths, structures of the termini, the identity of nucleotides adjacent to and near the loop, and the position of the guide (antisense) strand all affect the efficacy of shRNAs. In addition, shRNAs with 19-bp or shorter stem lengths are processed and function differently than those with longer stems. In this review, we describe studies of targeting the hepatitis C virus that have provided guidelines for an optimal design for short (19 bp) shRNAs (sshRNAs) that are highly potent, stable in biological fluids, and have minimal immunostimulatory properties.

Hepatitis C virus (HCV) is a major health concern with almost 3% of the world's population (350 million individuals) and 10% of the Pakistani population chronically infected with this viral pathogen. The current therapy of interferon-α and ribavirin against HCV has limited efficiency, so alternative options are desperately needed. RNA interference (RNAi), which results in a sequence-specific degradation of HCV RNA has potential as a powerful alternative molecular therapeutic approach. Concerning viral entry, the HCV structural gene E2 is mainly involved in virus attachment to the host cell surface receptors i.e., CD81 tetraspanin, scavenger receptor class B type 1 (SR-B1), low density lipoprotein receptor (LDLR) and claudin1 (CLDN1).

In this report, we studied the relationship of the HCV receptors CD81, LDL, CLDN1 and SR-B1to HCV infection. The potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was demonstrated by treatment with siRNAs against HCV receptors, which resulted in a significant decrease in HCV viral copy number.

Our data clearly demonstrate that the RNAi-mediated silencing of HCV receptors is among the first of its type for the development of an effective siRNA-based therapeutic option against HCV-3a. These findings will shed further light on the possible role of receptors in inhibition of HCV-3a viral titre through siRNA mediated silencing.

Hepatitis C virus (HCV) is a major threat as almost 3% of the world's population (350 million individual) and 10% of the Pakistani population is chronically infected with this virus. RNA interference (RNAi), a sequence-specific degradation process of RNA, has potential to be used as a powerful alternative molecular therapeutic approach in spite of the current therapy of interferon-α and ribavirin against HCV which has limited efficiency. HCV structural gene E2 is mainly involved in viral cell entry via attachment with the host cell surface receptors i.e., CD81 tetraspanin, low density lipoprotein receptor (LDLR), scavenger receptor class B type 1 (SR-B1), and Claudin1 (CLDN1). Considering the importance of HCV E2 gene and cellular receptors in virus infection and silencing effects of RNAi, the current study was designed to target the cellular and viral factors as new therapeutic options in limiting HCV infection.

In this study the potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was investigated by combined treatment of siRNAs against the HCV E2 gene and HCV cellular receptors (CD81 and LDLR), which resulted in a significant decrease in HCV viral copy number.

From the current study it is concluded that the combined RNAi-mediated silencing of HCV E2 and HCV receptors is important for the development of effective siRNA-based therapeutic option against HCV-3a.

The genus Nairovirus in the family Bunyaviridae contains 34 tick-borne viruses classified into seven serogroups. Hazara virus (HAZV) belongs to the Crimean-Congo hemorrhagic fever (CCHF) serogroup that also includes CCHF virus (CCHFV) a major pathogen for humans. HAZV is an interesting model to study CCHFV due to a close serological and phylogenetical relationship and a classification which allows handling in a BSL2 laboratory. Nairoviruses are characterized by a tripartite negative-sense single stranded RNA genome (named L, M and S segments) that encode the RNA polymerase, the Gn-Gc glycoproteins and the nucleoprotein (NP), respectively. Currently, there are neither vaccines nor effective therapies for the treatment of any bunyavirus infection in humans. In this study we report, for the first time, the use of RNA interference (RNAi) as an approach to inhibit nairovirus replication.

Chemically synthesized siRNAs were designed to target the mRNA produced by the three genomic segments. We first demonstrated that the siRNAs targeting the NP mRNA displayed a stronger antiviral effect than those complementary to the L and M transcripts in A549 cells. We further characterized the two most efficient siRNAs showing, that the induced inhibition is specific and associated with a decrease in NP synthesis during HAZV infection. Furthermore, both siRNAs depicted an antiviral activity when used before and after HAZV infection. We next showed that HAZV was sensitive to ribavirin which is also known to inhibit CCHFV. Finally, we demonstrated the additive or synergistic antiviral effect of siRNAs used in combination with ribavirin.

Our study highlights the interest of using RNAi (alone or in combination with ribavirin) to treat nairovirus infection. This approach has to be considered for the development of future antiviral compounds targeting CCHFV, the most pathogenic nairovirus.

Hepatitis C virus (HCV) is a major causative agent of liver associated diseases leading to the development of hepatocellular carcinoma (HCC) all over the world and genotype-3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current chemotherapy of interferon-α (IFN-α) and ribavirin against HCV infection alternative options are desperately needed out of which the recently discovered RNAi represent a powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process to silence virus infection or replication. HCV translation is mediated by a highly conserved internal ribosome entry site (IRES) within the 5'UTR region making it a relevant target for new drug development.

The present study was proposed to assess and explore the possibility of HCV silencing using siRNA targeting 5'UTR. For this analysis full length HCV 5'UTR of HCV-3a (pCR3.1/5'UTR) was tagged with GFP protein for in vitro analysis in Huh-7 cells. siRNA targeting 5'UTR were designed, and tested against constructed vector in Huh-7 cell line both at RNA and Protein levels. Furthermore, the effect of these siRNAs was confirmed in HCV-3a serum infected Huh-7 cell line.

The expression of 5'UTR-GFP was dramatically reduced both at mRNA and protein levels as compared with Mock transfected and control siRNAs treated cells using siRNAs against IRES of HCV-3a genotype. The potential of siRNAs specificity to inhibit HCV-3a replication in serum-infected Huh-7 cells was also investigated; upon treatment with siRNAs a significant decrease in HCV viral copy number and protein expression was observed.

Overall, the present work of siRNAs against HCV 5'UTR inhibits HCV-3a expression and represents effective future therapeutic opportunities against HCV-3a genotype.

Hepatitis C virus (HCV), a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Currently, the only treatment available consists of a combination of Pegylated interferon alpha (INF-α) and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi) represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV infection.

This study was design to assess or explore the silencing effect of small interference RNAs (siRNAs) against full length HCV particles of genotype 1a. In the present study six 21-bp siRNAs were designed against different regions of HCV structural genes (Core, E1 and E2). Selected siRNAs were labeled as Csi 301, Csi 29, E1si 52, E1si 192, E2si 86 and E2si 493. Our results demonstrated that siRNAs directed against HCV core gene showed 70% reduction in viral titer in HCV infected liver cells. Moreover, siRNAs against E1 and E2 envelop genes showed a dramatic reduction in HCV viral RNA, E2si 86 exhibited 93% inhibition, while E1si 192, E2si 493 and E1si 52 showed 87%, 80%, and 66% inhibition respectively. No significant inhibition was detected in cells transfected with the negative control siRNA.

Our results suggested that siRNAs targeted against HCV structural genes efficiently silence full length HCV particles and provide an effective therapeutic option against HCV infection.

Hepatitis C Virus (HCV) infection is a major health problem throughout world that causes acute and chronic infection which resulted in liver fibrosis, hepatocellular carcinoma and death. The only therapy currently available for HCV infection is the combination of pegylated interferon alpha (PEG-IFN α) and ribavirin. This therapy can effectively clear the virus infection in only 50% of infected individuals. Hence, there is a dire need to develop antiviral agents against HCV.

This study was design to examine the ability of exogenous small interfering RNAs (siRNAs) to block the replication of HCV in human liver cells. In the present study six 21-bp siRNAs were designed against different regions of HCV non-structural genes (NS2, NS3 serine protease/helicase, NS4Band NS5B RNA dependent RNA polymerase). siRNAs were labeled as NS2si241, NS3si-229, NS3si-858, NS4Bsi-166, NS5Bsi-241 and NS5Bsi-1064. We found that siRNAs against HCV NS2- NS5B efficiently inhibit HCV replication in Huh-7 cells. Our results demonstrated that siRNAs directed against HCV NS3 (NS3si-229 and NS3si-858) showed 58% and 88% reduction in viral titer respectively. Moreover, NS4Bsi-166 and NS5Bsi-1064 exhibited a dramatic reduction in HCV viral RNA and resulted in greater than 90% inhibition at a 20 μM concentration, while NS2si-241 showed 27% reduction in viral titer. No significant inhibition was detected in cells transfected with the negative control siRNA.

Our results suggest that siRNAs targeting against HCV non-structural genes (NS2-NS5B) efficiently inhibit HCV replication and combination of these siRNAs of different targets and interferon will be better option to treat HCV infection throughout the world.

Hepatitis C virus (HCV) is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option.

The current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs) against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed.

Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.

• Base Sequence
• Casein Kinase Ialpha/antagonists & inhibitors
• Casein Kinase Ialpha/genetics
• Cell Line
• DNA Primers/genetics
• Gene Library
• Gene Silencing
• Hepacivirus/genetics
• Hepacivirus/pathogenicity
• Hepacivirus/physiology
• Host-Pathogen Interactions/genetics
• Host-Pathogen Interactions/physiology
• Humans
• RNA, Small Interfering/genetics
• RNA, Viral/biosynthesis
• RNA, Viral/genetics
• Replicon
• Transfection
• Virus Replication/genetics
• Virus Replication/physiology

• MC_U130184143 Medical Research Council
• MC_UU_12014/1 Medical Research Council

The discovery and characterization of the RNA interference (RNAi) pathway has been one of the most important scientific developments of the last 12 years. RNAi is a cellular pathway wherein small RNAs control the expression of genes by either degrading homologous RNAs or preventing the translation of RNAs with partial homology. It has impacted basic biology on two major fronts. The first is the discovery of microRNAs (miRNAs), which regulate almost every cellular process and are required for some viral infections, including hepatitis C virus (HCV). The second front is the use of small interfering RNAs (siRNAs) as the first robust tool for mammalian cellular genetics. This has led to the identification of hundreds of cellular genes that are important for HCV infection. There is now a major push to adapt RNAi technology to the clinic. In this review, we explore the impact of RNAi in understanding HCV biology, the progress in design of RNAi-based therapeutics for HCV, and remaining obstacles.

• RNA interference
• hepatitis C virus
• siRNA therapy

• R01 AI080703 NIAID NIH HHS
• R56 AI080703 NIAID NIH HHS

• Antiviral Agents/pharmacology
• Catalytic Domain
• Computational Biology/methods
• Genome, Viral
• Genotype
• Hepacivirus/genetics
• Hepatitis C/transmission
• Hepatitis C/virology
• Lentivirus/genetics
• Models, Genetic
• RNA/metabolism
• RNA Interference
• RNA, Catalytic/chemistry
• RNA, Catalytic/genetics
• RNA, Viral/genetics
• Ribonuclease H/metabolism
• Transcription, Genetic

• 38322 CIHR

Hepatitis C virus (HCV) is one of the main causes of liver-related morbidity and mortality. Although combined interferon-α-ribavirin therapy is effective for about 50% of the patients with HCV, better therapies are needed and preventative vaccines have yet to be developed. Short-hairpin RNAs (shRNAs) inhibit gene expression by RNA interference. The application of transient shRNA expression is limited, however, due to the inability of the shRNA to replicate in mammalian cells and its inefficient transduction. The duration of transgene (shRNA) expression in mammalian cells can be significantly extended using baculovirus-based shRNA-expressing vectors that contain the latent viral protein Epstein-Barr nuclear antigen 1 (EBNA1) and the origin of latent viral DNA replication (OriP) sequences. These recombinant vectors contain compatible promoters and are highly effective for infecting primary hepatocyte and hepatoma cell lines, making them very useful tools for studies of hepatitis B and hepatitis C viruses. Here, we report the use of these baculovirus-based vector-derived shRNAs to inhibit core-protein expression in full-length hepatitis C virus (HCV) replicon cells.

We constructed a long-term transgene shRNA expression vector that contains the EBV EBNA1 and OriP sequences. We also designed baculovirus vector-mediated shRNAs against the highly conserved core-protein region of HCV. HCV core protein expression was inhibited by the EBNA1/OriP baculovirus vector for at least 14 days, which was considerably longer than the 3 days of inhibition produced by the wild-type baculovirus vector.

These findings indicate that we successfully constructed a long-term transgene (shRNA) expression vector (Ac-EP-shRNA452) using the EBNA1/OriP system, which was propagated in Escherichia coli and converted into mammalian cells. The potential anti-HCV activity of the long-term transgene (shRNA) expression vector was evaluated with the view of establishing highly effective therapeutic agents that can be further developed for HCV gene therapy applications.

• adenovirus vector
• hepatitis c virus
• rna interference

• Adenoviridae/genetics
• Animals
• Cell Line, Tumor
• Disease Models, Animal
• Feasibility Studies
• Genetic Therapy/methods
• Genetic Vectors
• Hepacivirus/genetics
• Hepacivirus/growth & development
• Hepacivirus/metabolism
• Hepatitis C/genetics
• Hepatitis C/metabolism
• Hepatitis C/prevention & control
• Humans
• Interferon Regulatory Factors/genetics
• Liver/metabolism
• Liver/virology
• Mice
• Mice, Inbred BALB C
• Mice, Transgenic
• RNA Interference
• RNA, Small Interfering/metabolism
• RNA, Viral
• Retroviridae/genetics
• Time Factors
• Transduction, Genetic
• Transfection
• Viral Proteins/biosynthesis
• Viral Proteins/genetics
• Virus Replication

• Animals
• Cations
• Drug Carriers/chemistry
• Drug Delivery Systems/methods
• Gene Silencing/drug effects
• Genome, Viral/drug effects
• Hepacivirus/drug effects
• Hepacivirus/genetics
• Lactose
• Liposomes/therapeutic use
• Liver/metabolism
• Liver/virology
• Mice
• Mice, Transgenic
• RNA, Small Interfering/administration & dosage
• RNA, Small Interfering/pharmacokinetics
• RNA, Small Interfering/pharmacology
• Treatment Outcome

• Gene Expression Regulation, Viral
• Hepacivirus/genetics
• Hepacivirus/physiology
• Hepatitis C, Chronic/therapy
• RNA Interference/physiology
• RNA, Small Interfering/physiology
• Replicon/genetics
• Virus Replication/physiology

• NIDDK-42182 PHS HHS

RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.

• RNA interference
• Short hairpin RNA
• Micro RNA
• Antiviral therapy
• Viral infection
• Human immunodeficiency virus
• Hepatitis C virus
• Hepatitis B virus
• SARS-coronavirus

• Antiviral Agents/therapeutic use
• HIV Infections/drug therapy
• HIV Infections/genetics
• Hepatitis B/drug therapy
• Hepatitis B/genetics
• Hepatitis C/drug therapy
• Hepatitis C/genetics
• Humans
• RNA Interference
• Severe Acute Respiratory Syndrome/drug therapy
• Severe Acute Respiratory Syndrome/genetics
• Virus Diseases/drug therapy
• Virus Diseases/genetics

• Animals
• Genetic Vectors/genetics
• Hepacivirus/genetics
• Hepatitis C/therapy
• Humans
• Models, Biological
• Mutation
• RNA Interference
• RNA, Small Interfering/genetics
• Transfection/methods

• 5' Untranslated Regions
• Base Sequence
• Cell Line
• Gene Expression
• Genetic Vectors
• Hepacivirus/genetics
• Hepacivirus/metabolism
• Humans
• Molecular Sequence Data
• Nucleic Acid Conformation
• RNA Interference
• RNA, Small Interfering/chemistry
• RNA, Small Interfering/genetics
• RNA, Small Interfering/metabolism
• RNA, Viral/genetics
• Transfection

• N01-AI-30046 NIAID NIH HHS
• R43AI056611 NIAID NIH HHS
• R44AI056611 NIAID NIH HHS

• 5' Untranslated Regions/genetics
• Animals
• Base Sequence
• Callithrix
• Disease Models, Animal
• GB virus B/genetics
• GB virus B/physiology
• Genes, Reporter
• Hepatitis C/virology
• Interferons/blood
• Liposomes
• Male
• Mice
• Molecular Sequence Data
• Monkey Diseases/virology
• Nucleic Acid Conformation
• RNA, Small Interfering/metabolism
• RNA, Viral/chemistry
• RNA, Viral/genetics
• Virus Replication/physiology

• Drug Design
• Gene Expression Profiling/methods
• Gene Expression Regulation, Viral/genetics
• Gene Library
• Genetic Testing/methods
• Hepacivirus/genetics
• Hepacivirus/growth & development
• Hepatitis C, Chronic/genetics
• Hepatitis C, Chronic/virology
• Humans
• Interferons/immunology
• RNA, Small Interfering
• RNA, Viral/genetics
• Replicon/genetics
• Virus Replication/genetics

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and hepatocellular carcinoma, yet fully efficacious treatments are missing. In this study, we investigated RNA interference (RNAi), a specific gene silencing process mediated by small interfering RNA (siRNA) duplexes, as an antiviral strategy against HCV. Synthetic siRNAs were designed to target conserved sequences of the HCV 5′ nontranslated region (NTR) located in a functional, stem–loop structured domain of the HCV internal ribosome entry site (IRES), which is crucial for initiation of polyprotein translation. Several siRNAs dramatically reduced or even abrogated the replication of selectable subgenomic HCV replicons upon cotransfection of human hepatoma cells with viral target and siRNAs, or upon transfection of cells supporting autonomous replication of HCV replicon with siRNAs. Importantly, three siRNAs also proved capable of strongly inhibiting virus production in cell culture. One siRNA, targeting a sequence that is highly conserved across all genotypes and forms a critical pseudoknot structure involved in translation, was identified as the most promising therapeutic candidate. These results indicate that the HCV life cycle can be efficiently blocked by using properly-designed siRNAs that target functionally important, highly conserved sequences of the HCV IRES. This finding offers a novel approach towards developing IRES-based antiviral treatment for chronic HCV infections.

• Base Sequence
• Cell Line, Tumor
• DNA Replication
• Genome, Viral/genetics
• Hepacivirus/physiology
• Humans
• Molecular Sequence Data
• Nucleic Acid Conformation
• RNA, Small Interfering/genetics
• RNA, Untranslated/chemistry
• RNA, Untranslated/genetics
• Replicon/genetics

• R24 RR015081 NCRR NIH HHS
• R24-RR15081 NCRR NIH HHS

• Animals
• Hepacivirus/physiology
• Hepatitis C/physiopathology
• Hepatitis C/therapy
• Humans

• 3' Untranslated Regions
• Antiviral Agents/pharmacology
• Cell Line
• Genes, Reporter
• Hepacivirus/genetics
• Hepacivirus/physiology
• Humans
• Luciferases/analysis
• Luciferases/genetics
• RNA Interference
• RNA, Small Interfering/genetics
• RNA, Small Interfering/pharmacology
• RNA, Viral/genetics
• RNA, Viral/metabolism

• DK42182 NIDDK NIH HHS

• Antiviral Agents/administration & dosage
• Antiviral Agents/adverse effects
• Antiviral Agents/pharmacology
• Antiviral Agents/therapeutic use
• Drug Therapy, Combination
• Hepacivirus/drug effects
• Hepacivirus/immunology
• Hepatitis C/drug therapy
• Hepatitis C/prevention & control
• Humans
• Interferons/therapeutic use
• Ribavirin/pharmacology
• Ribavirin/therapeutic use
• Viral Hepatitis Vaccines/therapeutic use

• Animals
• Antiviral Agents
• Base Sequence
• Bovine Virus Diarrhea-Mucosal Disease/immunology
• Bovine Virus Diarrhea-Mucosal Disease/prevention & control
• Bovine Virus Diarrhea-Mucosal Disease/virology
• Cattle
• Cell Line
• Diarrhea Viruses, Bovine Viral/genetics
• Diarrhea Viruses, Bovine Viral/physiology
• Gene Silencing
• Genetic Therapy/methods
• Genetic Therapy/veterinary
• Genetic Vectors
• Molecular Sequence Data
• RNA Interference
• RNA, Small Interfering/physiology
• RNA, Viral/biosynthesis
• Transfection
• Virus Replication/genetics
• Virus Replication/immunology