A comparison of multiple shRNA expression methods for combinatorial RNAi
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SHORT REPORT
GENETIC VACCINES AND THERAPY
Open Access
A comparison of multiple shRNA expression methods for combinatorial RNAi Glen J Mcintyre1,2*, Allison J Arndt1, Kirsten M Gillespie1, Wendy M Mak1 and Gregory C Fanning1,3
Abstract 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. Introduction The recently discovered RNA interference (RNAi) pathway is a post-transcriptional gene silencing and regulation mechanism with potential application in the field of gene therapy. In mammalian cells RNAi begins with a double-stranded RNA inducer that is progressively processed from its termini by RNase III type endonucleases, firstly Drosha in the nucleus followed by Dicer in the cytoplasm, to yield a short interfering RNA (siRNA) duplex of ~ 22 bp [1,2]. The duplex is unwound and loaded into the RNA induced silencing complex (RISC) in a process that favors one of the two strands (the guide strand) based on a difference in thermodynamic stability at the ends of the duplex [3]. The most common natural substrates for mammalian RNAi are microRNA, short hairpin-like RNA transcripts implicated in regulating gene expression activity [1,2]. The RNAi pathway can be artificially engaged at any point in the process, typically either through delivering synthetic siRNAs to the RISC [4,5] or by expressing short hairpin RNAs (shRNA or
* Correspondence: [email protected] 1 Johnson and Johnson Research Pty Ltd, Level 4 Biomedical Building, 1 Central Avenue, Australian Technology Park, Eveleigh, NSW, 1430, Australia Full list of author information is available at the end of the article
hairpins) to be processed by Dicer and possibly Drosha [6,7]. shRNAs are well suited for use in current gene therapy plans. shRNA consists of a short single-stranded RNA transcript that folds into a ‘hairpin’ configuration by virtue of self-complementary regions separated b
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