Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

Liu, Ying Poi; Vink, Monique; Westerink, Jan-Tinus; Arellano, Eva Ramírez de; Konstantinova, Pavlina; Brake, Olivier ter; Berkhout, Ben

doi:10.1261/rna.1887910

Cited by 64 publications

(56 citation statements)

References 57 publications

(65 reference statements)

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“…ed titer problems with lentiviral vectors encoding antiviral shRNAs that may obstruct clinical application [44][45][46][47][48] . Lentiviral vectors are produced by co-transfection of the lentiviral vector construct JS1 ( Figure 2B), Gag-Pol and Revexpression plasmids, and a VSV-G envelope construct.…”

Section: Culturesmentioning

confidence: 99%

“…This is referred to as vector targeting. We previously discussed in detail all possible routes by which shRNAs could impede lentiviral vector production and how to prevent or overcome these specific problems [45,48] . Of course, acute cytotoxicity of the expressed shRNA can also cause a serious titer reduction due to effects on the producer cell viability and this may eventually also affect the viability of transduced cells, i.e., the gene therapy target cells.…”

Section: Culturesmentioning

confidence: 99%

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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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Section: Culturesmentioning

confidence: 99%

Section: Culturesmentioning

confidence: 99%

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

Knoepfel

Centlivre²,

Liu³

et al. 2012

WJV

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“…This has the potential to result in a reduction in viral replication that would potentially exceed any enhancement caused by the viral miRNA, and this problem would become more extreme if several viral pre-miRNAs were excised from a single viral genome. Indeed, the ability of inserted pri-miRNA stem-loops to reduce viral titers in cis has been well documented in the case of retroviral vectors, although this reduction can largely be alleviated by knockdown of Drosha in the producer cells (9,10).…”

mentioning

confidence: 99%

Identification of Novel, Highly Expressed Retroviral MicroRNAs in Cells Infected by Bovine Foamy Virus

Whisnant

Kehl

Bao

et al. 2014

J Virol

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While numerous viral microRNAs (miRNAs) expressed by DNA viruses, especially herpesvirus family members, have been reported, there have been very few reports of miRNAs derived from RNA viruses. Here we describe three miRNAs expressed by bovine foamy virus (BFV), a member of the spumavirus subfamily of retroviruses, in both BFV-infected cultured cells and BFV-infected cattle. All three viral miRNAs are initially expressed in the form of an ϳ122-nucleotide (nt) pri-miRNA, encoded within the BFV long terminal repeat U3 region, that is subsequently cleaved to generate two pre-miRNAs that are then processed to yield three distinct, biologically active miRNAs. The BFV pri-miRNA is transcribed by RNA polymerase III, and the three resultant mature miRNAs were found to contribute a remarkable ϳ70% of all miRNAs expressed in BFV-infected cells. These data document the second example of a retrovirus that is able to express viral miRNAs by using embedded proviral RNA polymerase III promoters. IMPORTANCE Foamy viruses are a ubiquitous family of nonpathogenic retroviruses that have potential as gene therapy vectors in humans.Here we demonstrate that bovine foamy virus (BFV) expresses high levels of three viral microRNAs (miRNAs) in BFV-infected cells in culture and also in infected cattle. The BFV miRNAs are unusual in that they are initially transcribed by RNA polymerase III as a single, ϳ122-nt pri-miRNA that is subsequently processed to release three fully functional miRNAs. The observation that BFV, a foamy virus, is able to express viral miRNAs in infected cells adds to emerging evidence that miRNA expression is a common, albeit clearly not universal, property of retroviruses and suggests that these miRNAs may exert a significant effect on viral replication in vivo. M icroRNAs (miRNAs) are a diverse class of ϳ22-nucleotide (nt) regulatory RNAs that are expressed by all known multicellular eukaryotes (1). Cellular miRNAs are normally transcribed by RNA polymerase II (Pol II) to give rise to a long primiRNA that is cleaved in the nucleus by the microprocessor complex, consisting of the RNase III enzyme Drosha and the RNA-binding cofactor DGCR8, to release the ϳ60-nt pre-miRNA intermediate, which contains an ϳ22-bp imperfect stem with an ϳ2-nt 3= overhang (2). The pre-miRNA is then exported to the cytoplasm, where it is cleaved by a second RNase III enzyme, Dicer, which removes the terminal loop, leaving a second ϳ2-nt 3= overhang.

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“…It was demonstrated that knockdown of Drosha can indeed increase the titer of such vectors. 186,187 When comparing a large series of LV constructs, a dramatically low titer was measured for LVs that express one or multiple artificial miRNA(s), but this reduction was largely due to the internal Pol II promoter element. 186 The titer could be restored almost completely by deletion or replacement of this promoter.…”

Section: Vector Production Issuesmentioning

confidence: 99%

“…186,187 When comparing a large series of LV constructs, a dramatically low titer was measured for LVs that express one or multiple artificial miRNA(s), but this reduction was largely due to the internal Pol II promoter element. 186 The titer could be restored almost completely by deletion or replacement of this promoter. It is possible that transgene expression from the CMV promoter is favored over RSV promoter-driven expression of the vector RNA genome (Fig.…”

Section: Vector Production Issuesmentioning

confidence: 99%

Improving miRNA Delivery by Optimizing miRNA Expression Cassettes in Diverse Virus Vectors

Herrera-Carrillo

Liu

Berkhout

2017

Human Gene Therapy Methods

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The RNA interference pathway is an evolutionary conserved post-transcriptional gene regulation mechanism that is exclusively triggered by double-stranded RNA inducers. RNAi-based methods and technologies have facilitated the discovery of many basic science findings and spurred the development of novel RNA therapeutics. Transient induction of RNAi via transfection of synthetic small interfering RNAs can trigger the selective knockdown of a target mRNA. For durable silencing of gene expression, either artificial short hairpin RNA or microRNA encoding transgene constructs were developed. These miRNAs are based on the molecules that induce the natural RNAi pathway in mammals and humans: the endogenously expressed miRNAs. Significant efforts focused on the construction and delivery of miRNA cassettes in order to solve basic biology questions or to design new therapy strategies. Several viral vectors have been developed, which are particularly useful for the delivery of miRNA expression cassettes to specific target cells. Each vector system has its own unique set of distinct properties. Thus, depending on the specific application, a particular vector may be most suitable. This field was previously reviewed for different viral vector systems, and now the recent progress in the field of miRNA-based gene-silencing approaches using lentiviral vectors is reported. The focus is on the unique properties and respective limitations of the available vector systems for miRNA delivery.Keywords: miRNA cassettes, gene therapy, viral vectors, vector tropism INTRODUCTION NON-CODING RNA MOLECULES play an essential role in gene regulation of the cell via a mechanism called RNA interference (RNAi). The RNAi mechanism is evolutionary conserved in eukaryotes and triggers the sequence-specific inhibition or degradation of a single or a unique set of complementary mRNAs. Three small RNA classes have been described to participate in mammalian RNAi mechanisms: microRNAs (miRNAs), 1,2 endogenous small interfering RNAs (endo-siRNAs), 3 and PIWI-associated RNAs (piRNAs). 4 The endo-siRNAs and piRNAs are involved in suppression of transposable elements. The miRNAs are broadly involved in the regulated expression of multiple cellular genes and execute their effect at the posttranscriptional level. 5,6 MiRNA-mediated regulation of gene expression plays a significant role in cell metabolism and cellular developmental and differentiation processes in mammals. More than a thousand human miRNAs have already been identified that are involved in the regulated expression of around 30% of all genes, which is a conservative estimate.7 Because of the miRNA abundance and their important regulatory functions, these molecules have received much attention from the scientific community over the last decade. For instance, efforts have been made to explain the biological function of the natural miRNAs by identifying the target mRNA and the role in cellular physiology. On the other hand, the design of man-made miRNA mimics to impose control over gene expression beca...

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Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

Cited by 64 publications

References 57 publications

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

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

Identification of Novel, Highly Expressed Retroviral MicroRNAs in Cells Infected by Bovine Foamy Virus

Improving miRNA Delivery by Optimizing miRNA Expression Cassettes in Diverse Virus Vectors

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