title: A comparison of multiple shRNA expression methods for combinatorial RNAi.
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.
authors.affiliation: Johnson and Johnson Research Pty Ltd, Level 4 Biomedical Building, 1 Central Avenue, Australian Technology Park, Eveleigh, NSW, 1430, Australia. glen@madebyglen.com.
authors.forename: Glen J
authors.initials: GJ
authors.lastname: McIntyre
authors.forename: Allison J
authors.initials: AJ
authors.lastname: Arndt
authors.forename: Kirsten M
authors.initials: KM
authors.lastname: Gillespie
authors.forename: Wendy M
authors.initials: WM
authors.lastname: Mak
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Fanning