Combinatorial RNAi Against HIV-1 Using Extended Short Hairpin RNAs

Liu, Ying Poi; Eije, Karin J. von; Schopman, Nick C.T.; Westerink, Jan-Tinus; Brake, Olivier ter; Haasnoot, Joost; Berkhout, Ben

doi:10.1038/mt.2009.176

Cited by 85 publications

(91 citation statements)

References 47 publications

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“…Using two cell-culture models for viral infection, we validated the top-scoring shRNAs and demonstrated their potent antiviral effect. These shRNAs target various genes of the viral genomes; therefore they could be tested for combinatorial treatment of viral infection (21,22). Particularly, shRNAs targeting HCV demonstrate the ability to almost completely inhibit viral infectivity.…”

Section: Discussionmentioning

confidence: 99%

Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy

Tan¹,

Gao³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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shRNAs can trigger effective silencing of gene expression in mammalian cells, thereby providing powerful tools for genetic studies, as well as potential therapeutic strategies. Specific shRNAs can interfere with the replication of pathogenic viruses and are currently being tested as antiviral therapies in clinical trials. However, this effort is hindered by our inability to systematically and accurately identify potent shRNAs for viral genomes. Here we apply a recently developed highly parallel sensor assay to identify potent shRNAs for HIV, hepatitis C virus (HCV), and influenza. We observe known and previously unknown sequence features that dictate shRNAs efficiency. Validation using HIV and HCV cell culture models demonstrates very high potency of the top-scoring shRNAs. Comparing our data with the secondary structure of HIV shows that shRNA efficacy is strongly affected by the secondary structure at the target RNA site. Artificially introducing secondary structure to the target site markedly reduces shRNA silencing. In addition, we observe that HCV has distinct sequence features that bias HCV-targeting shRNAs toward lower efficacy. Our results facilitate further development of shRNA based antiviral therapies and improve our understanding and ability to predict efficient shRNAs.

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Section: Discussionmentioning

confidence: 99%

Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy

Tan¹,

Gao³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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%

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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“…Indeed, different synthetic molecules targeting specific RNA motifs essential for viral RNA multiplication have been exploited as therapeutic agents. [46][47][48][49][50][51] Furthermore, the IRES-inhibitory property of IRAB was observed both in in vitro translation assays using cell free lysates and in cells transfected with infectious RNA revealing that the primary targeted event is viral RNA translation, which is IRES-dependent. 7 In agreement with this, a specific decrease of viral protein synthesis occurred in transfected cells concomitantly to a reduction in virus yield in the presence of IRAB (2 mM).…”

Section: Discussionmentioning

confidence: 99%

“…The EC 50 of SL3A/AP167 C tRNA and SLIIa/AP54 C tRNA were determined by performing fluorescence titrations in the presence of a 10-fold molar excess of competitor tRNA mix . The later data were used to calculate the selectivities of the ligands for the given oligonucleotide, expressed as the ratio of [EC 50 with tRNA] to [EC 50 without tRNA].…”

Section: Discussionmentioning

confidence: 99%

“…The EC 50 determined for the mixtures of tRNA and either SL3A/AP167 or SLIIa/AP54 are higher than those found for the oligonucleotides alone, as often in this kind of competition experiments. 25 From these data, the degree of selectivity of the ligands for their respective targets can be calculated as the ratio of [EC 50 with tRNA] to [EC 50 without tRNA] (Fig. 6D).…”

Section: Irab Induces a Local Structural Reorganization Of The Ires Ementioning

confidence: 99%

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Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation

et al. 2015

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(2015) Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation, RNA Biology, 12:5, 555-568, DOI: 10.1080/15476286.2015 The internal ribosome entry site (IRES) element located at the 5´untranslated genomic region of various RNA viruses mediates cap-independent initiation of translation. Picornavirus IRES activity is highly dependent on both its structural organization and its interaction with host factors. Small molecules able to interfere with RNA function are valuable candidates for antiviral agents. Here we show that a small molecule based on benzimidazole (IRAB) inhibited foot-andmouth disease virus (FMDV) IRES-dependent protein synthesis in cells transfected with infectious RNA leading to a decrease of the virus titer, which was higher than that induced by a structurally related benzimidazole derivative. Interestingly, IRAB preferentially inhibited IRES-dependent translation in cell free systems in a dose-dependent manner. RNA structural analysis by SHAPE demonstrated an increased local flexibility of the IRES structure upon incubation with IRAB, which affected 3 stem-loops (SL) of domain 3. Fluorescence binding assays conducted with individual aminopurinelabeled oligoribonucleotides indicated that the SL3A binds IRAB (EC 50 18 mM). Taken together, the results derived from SHAPE reactivity and fluorescence binding assays suggested that the target site of IRAB within the FMDV IRES might be a folded RNA structure that involves the entire apical region of domain 3. Our data suggest that the conformational changes induced by this compound on a specific region of the IRES structure which is essential for its activity is, at least in part, responsible for the reduced IRES efficiency observed in cell free lysates and, particularly, in RNA-transfected cells.

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Combinatorial RNAi Against HIV-1 Using Extended Short Hairpin RNAs

Cited by 85 publications

References 47 publications

Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy

Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy

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

Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation

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