Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron

Liu, Ying Poi; Haasnoot, Joost; Brake, Olivier ter; Berkhout, Ben; Konstantinova, Pavlina

doi:10.1093/nar/gkn109

Cited by 187 publications

(184 citation statements)

References 66 publications

(84 reference statements)

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“…1A) 15 to develop a multiplexed platform for inhibiting HCV, similar to one designed to inhibit HIV. 16 A liver-specific promoter was used to ensure expression in hepatocytes. The HCV target sequences, their location in HCV 1b, the names of the miRNAs designed to cleave them, and the miRNAs they replace in the endogenous miR-17-92 cluster are shown in Table 1.…”

Section: Resultsmentioning

confidence: 99%

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Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microrna cluster

et al. 2010

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RNA interference (RNAi) is being evaluated as an alternative therapeutic strategy for hepatitis C virus (HCV) infection. The use of viral vectors encoding short hairpin RNAs (shRNAs) has been the most common strategy employed to provide sustained expression of RNAi effectors. However, overexpression and incomplete processing of shRNAs has led to saturation of the endogenous miRNA pathway, resulting in toxicity. The use of endogenous microRNAs (miRNAs) as scaffolds for short interfering (siRNAs) may avoid these problems, and miRNA clusters can be engineered to express multiple RNAi effectors, a feature that may prevent RNAi-resistant HCV mutant generation. We exploited the endogenous miRNA-17-92 cluster to generate a polycistronic primary miRNA that is processed into five mature miRNAs that target different regions of the HCV genome. All five anti-HCV miRNAs were active, achieving up to 97% inhibition of Renilla luciferase (RLuc) HCV reporter plasmids. Self-complementary recombinant adeno-associated virus (scAAV) vectors were chosen for therapeutic delivery of the miRNA cluster. Expression of the miRNAs from scAAV inhibited the replication of cell culture-propagated HCV (HCVcc) by 98%, and resulted in up to 93% gene silencing of RLuc-HCV reporter plasmids in mouse liver. No hepatocellular toxicity was observed at scAAV doses as high as 5 3 10 11 vector genomes per mouse, a dose that is approximately five-fold higher than doses of scAAV-shRNA vectors that others have shown previously to be toxic in mouse liver. Conclusion: We have demonstrated that exogenous anti-HCV miRNAs induce gene silencing, and when expressed from scAAV vectors inhibit the replication of HCVcc without inducing toxicity. The combination of an AAV vector delivery system and exploitation of the endogenous RNAi pathway is a potentially viable alternative to current HCV treatment regimens. (HEPATOLOGY 2010;52:1877-1887 H epatitis C virus (HCV) infection remains a major worldwide health care problem, because approximately 3% of the world population is chronically infected with this virus, which causes viral hepatitis and can lead to cirrhosis and hepatocellular carcinoma.1 HCV replicates in the cytoplasm by a virally encoded RNA-dependent RNA polymerase (nonstructural protein 5B [NS5B]), and like most RNA polymerases, NS5B has low fidelity and incorporates mutations into its genome at a rate of $10 À4 base substitutions/nucleotide, 2 generating $one mutation per round of replication. Thus, HCV shows extraordinary genetic diversity with six major genotypes, at least 50 subtypes, and millions of quasispecies. This feature of HCV has made vaccine and drug development extremely challenging. Although HCV infections are currently managed with a combination of pegylated interferon-a and ribavirin, this regimen is successful in achieving a sustained virological response in only approximately 50% of patients infected with

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Section: Resultsmentioning

confidence: 99%

“…The endogenous miR-17-92 cluster 15,16 was modified by replacing the first five mature miRNAs of the cluster with inhibitory RNAs targeting HCV. All five miRNAs were effective in knocking down expression of Renilla luciferase (RLuc)-HCV reporter plasmids, both in vitro and in vivo, by up to 97%.…”

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confidence: 99%

Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microrna cluster

et al. 2010

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“…For several years, we have designed and tested various alternative RNAi strategies against HIV-1. Extended shRNA designs and miRNA-like polycistron transcripts were optimized for the expression of multiple inhibitors, but the use of independent shRNA cassettes turned out to be most efficient [9,14,[17][18][19][20][21] . Thus, the goal is to use a lentiviral vector with multiple shRNA cassettes that becomes stably incorporated in the human genome.…”

Section: Guidelines For Basic Sciencementioning

confidence: 99%

“…This combinatorial strategy is analogous to current antiretroviral therapy regimens with multiple drugs that have led to significant clinical success in HIV-1 infected patients [57] . There are several ways to express multiple RNAi inhibitors against HIV-1, ranging from polycistronic miRNAs to extended multimeric shRNA transcripts [17][18][19][20][21] . We achieved most promising results with multiple shRNA cassettes [14,16] .…”

Section: Combinatorial Rnaimentioning

confidence: 99%

Selection of RNAi-based inhibitors for anti-HIV gene therapy

Knoepfel

Centlivre²,

Liu³

et al. 2012

WJV

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In the last decade, RNA interference (RNAi) advanced to one of the most widely applied techniques in the biomedical research field and several RNAi therapeutic clinical trials have been launched. We focus on RNAibased inhibitors against the chronic infection with human immunodeficiency virus type 1 (HIV-1). A lentiviral gene therapy is proposed for HIV-infected patients that will protect and reconstitute the vital immune cell pool. The RNAi-based inhibitors that have been developed are short hairpin RNA molecules (shRNAs), of which multiple are needed to prevent viral escape. In ten distinct steps, we describe the selection process that started with 135 shRNA candidates, from the initial design criteria, via testing of the in vitro and in vivo antiviral activity and cytotoxicity to the final design of a combinatorial therapy with three shRNAs. These shRNAs satisfied all 10 selection criteria such as targeting conserved regions of the HIV-1 RNA genome, exhibiting robust inhibition of HIV-1 replication and having no impact on cell physiology. This combinatorial shRNA vector will soon move forward to the first clinical studies.

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“…A recent advance in gene silencing methodology is to replace the natural stem sequence of pri-miRNA with synthetic target-specific sequence. The mimics of pri-miRNA, shRNAmirs, serve as the natural substrates in miRNA biogenesis pathways and can trigger potent gene knockdown (Chung et al, 2006;Liu et al, 2008). Moreover, shRNAmir-based systems are less prone to cause toxicity by interfering with the endogenous miRNA pathway (Boudreau et al, 2009;McBride et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

RNA interference of influenza A virus replication by microRNA-adapted lentiviral loop short hairpin RNA

Liu

et al. 2015

Journal of General Virology

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Limitations of the current vaccines and antivirals against influenza A virus (IAV) pandemic underscore the urgent need for developing novel anti-influenza strategies. RNA interference (RNAi) induced by small interfering RNA (siRNA) has become a powerful new means to inhibit viral infection in a gene-specific manner. However, the efficacy of the siRNA delivery platform and the relatively high cost of administration have hindered widespread application of siRNA. In this study, we developed a microRNA (miRNA)-30-based lentivirus delivery system by embedding a synthetic short hairpin RNA (shRNA) stem into the context of endogenous precursor of miRNA-30 (shRNAmir) to express a silencer of the influenza gene. We showed that the miRNA-based lentivirus vector was able to express and process a single nucleoprotein (NP)-targeting shRNAmir, which could potently inhibit IAV replication. We further showed that miRNA-based lentivirus vector carrying tandemly linked NP and polymerase PB1 shRNAmirs could express and process double shRNAmirs. Despite the relatively low levels of NP and PB1 miRNAs produced in the stably transduced cells, the combination of two miRNAs exerted a great degree of inhibition on influenza infection. Given the advantage of combinatorial RNAi in preventing emergence of mutant virus, miRNA-based lentiviral vectors are valuable tools for anitiviral activities. To the best of our knowledge, this is the first study demonstrating that a miRNA-based RNAi strategy can be applied for better control of influenza virus infection.

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Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron

Cited by 187 publications

References 66 publications

Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microrna cluster

Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microrna cluster

Selection of RNAi-based inhibitors for anti-HIV gene therapy

RNA interference of influenza A virus replication by microRNA-adapted lentiviral loop short hairpin RNA

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