Cassette deletion in multiple shRNA lentiviral vectors for HIV-1 and its impact on treatment success

Mcintyre, Glen J; Yu, Yi Hsin; Tran, Anna H.; Jaramillo, Angel B.; Arndt, Allison J; Millington, Michelle; Boyd, Maureen P.; Elliott, Fiona; Shen, Sylvie; Murray, John M.; Applegate, Tanya

doi:10.1186/1743-422x-6-184

Cited by 19 publications

(19 citation statements)

References 56 publications

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“…It is worth noting that a similar study was published during the preparation of this manuscript, with similar conclusions, thus strengthening the validity of our findings [70]. Furthermore, we have since applied the multiple cassette strategy in several additional studies, including the development of a repeating modular cloning method (tested with up to 11 shRNAs), the assembly of combinations of up to 7 shRNAs to target entire subtypes of HIV-1, and a large-scale study around repeat-mediated deletion of 1 or more cassettes [21,71]. …”

Section: Discussionsupporting

confidence: 73%

A comparison of multiple shRNA expression methods for combinatorial RNAi

Mcintyre

Arndt

Gillespie

et al. 2011

Genet Vaccines Ther

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RNAi gene therapies for HIV-1 will likely need to employ multiple shRNAs to counter resistant strains. We evaluated 3 shRNA co-expression methods to determine their suitability for present use; multiple expression vectors, multiple expression cassettes and single transcripts comprised of several dsRNA units (aka domains) with each being designed to a different target. Though the multiple vector strategy was effective with 2 shRNAs, the increasing number of vectors required is a major shortcoming. With single transcript configurations we only saw adequate activity from 1 of 10 variants tested, the variants being comprised of 2 - 3 different target domains. Whilst single transcript configurations have the most advantages on paper, these configurations can not yet be rapidly and reliably re-configured for new targets. However, our multiple cassette combinations of 2, 3 and 4 (29 bp) shRNAs were all successful, with suitable activity maintained in all positions and net activities comparable to that of the corresponding single shRNAs. We conclude that the multiple cassette strategy is the most suitably developed for present use as it is easy to design, assemble, is directly compatible with pre-existing shRNA and can be easily expanded.

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Section: Discussionsupporting

confidence: 73%

A comparison of multiple shRNA expression methods for combinatorial RNAi

Mcintyre

Arndt

Gillespie

et al. 2011

Genet Vaccines Ther

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“…To express multiple shRNAs, we initially inserted several H1-driven shRNA expression cassettes into the lentiviral vector. However, these vectors are extremely unstable as shRNA cassettes were deleted during transduction due to slippage of the Reverse Transcriptase enzyme on the repeated H1 promoter sequences [16,58] . To prevent recombination-mediated deletion of shRNA cassettes, we designed shRNA cassettes with unique promoter elements.…”

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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“…The ability of the reverse transcriptase to switch between the two lentiviral RNA templates during reverse transcription makes direct repeat sequences prone to rearrangement by either duplication or deletion (Gilboa et al 1979;An and Telesnitsky 2001;ter Brake et al 2008;McIntyre et al 2009). To evaluate the integrity of the inhibitor cassette after lentiviral integration, we transduced cells at a very low MOI to obtain clones with single lentiviral integrations and performed PCR-based analyses of the inhibitor cassette on genomic DNA from pooled and single clones.…”

Section: Integrity Of the Inhibitor-encoding Cassettes During Lentivimentioning

confidence: 99%

Potent microRNA suppression by RNA Pol II-transcribed ‘Tough Decoy’ inhibitors

Bak

Hollensen

Primo

et al. 2012

RNA

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MicroRNAs (miRNAs) are key regulators of gene expression and modulators of diverse biological pathways. Analyses of miRNA function as well as therapeutic managing of miRNAs rely on cellular administration of miRNA inhibitors which may be achieved by the use of viral vehicles. This study explores the miRNA-suppressive capacity of inhibitors expressed intracellularly from lentivirusderived gene vectors. Superior activity of two decoy-type inhibitors, a "Bulged Sponge" with eight miRNA recognition sites and a hairpin-shaped "Tough Decoy" containing two miRNA recognition sites, is demonstrated in a side-by-side comparison of seven types of miRNA inhibitors transcribed as short RNAs from an RNA Pol III promoter. We find that lentiviral vectors expressing Tough Decoy inhibitors are less vulnerable than Bulged Sponge-encoding vectors to targeting by the cognate miRNA and less prone, therefore, to reductions in transfer efficiency. Importantly, it is demonstrated that Tough Decoy inhibitors retain their miRNA suppression capacity in the context of longer RNA transcripts expressed from an RNA Pol II promoter. Such RNA Pol II-transcribed Tough Decoy inhibitors are new tools in managing of miRNAs and may have potential for temporal and spatial regulation of miRNA activity as well as for therapeutic targeting of miRNAs that are aberrantly expressed in human disease.

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Cassette deletion in multiple shRNA lentiviral vectors for HIV-1 and its impact on treatment success

Cited by 19 publications

References 56 publications

A comparison of multiple shRNA expression methods for combinatorial RNAi

A comparison of multiple shRNA expression methods for combinatorial RNAi

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

Potent microRNA suppression by RNA Pol II-transcribed ‘Tough Decoy’ inhibitors

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