In-Plant Protection against Helicoverpa armigera by Production of Long hpRNA in Chloroplasts.

Expressing double-stranded RNA (dsRNA) in transgenic plants to silence essential genes within herbivorous pests is referred to as trans-kingdom RNA interference (TK-RNAi) and has emerged as a promising strategy for crop protection. However, the dicing of dsRNA into siRNAs by the plant's intrinsic RNAi machinery may reduce this pesticidal activity. Therefore, genetic constructs, encoding ∼200 nt duplex-stemmed-hairpin (hp) RNAs, targeting the acetylcholinesterase gene of the cotton bollworm, Helicoverpa armigera, were integrated into either the nuclear or the chloroplast genome of Nicotiana benthamiana. Undiced, full-length hpRNAs accumulated in transplastomic lines of N. benthamiana and conferred strong protection against H. armigera herbivory while the hpRNAs of nuclear-transformed plants were processed into siRNAs and gave more modest anti-feeding activity. This suggests that there is little or no RNAi machinery or activity in the chloroplast, that hpRNAs produced within this organelle do not enter the cytoplasm, and that oral delivery of chloroplast-packaged intact hpRNA is a more effective means of delivering TK-RNAi than using nuclear encoded hpRNAs. This contrasts with a recently reported correlation between siRNA expression and effectiveness of TK-RNAi targeting the chitinase gene of H. armigera, but is consistent with reports of efficient TK-RNAi by dsRNA generated in chloroplasts by converging promoters flanking a pest gene sequence and from very small (21 nt-stem) hpRNAs resembling artificial miRNAs. Here we demonstrate that hpRNAs, constructed along the conventional design principles of plant RNAi constructs but integrated into the chloroplast genome, are stable and effective over multiple generations, and hold the promise of providing durable pest resistance in crops.

The effects of stem length and core placement on shRNA activity.

BACKGROUND: Expressed short hairpin RNAs (shRNA) used in mammalian RNA interference (RNAi) are often designed around a specific short interfering RNA (siRNA) core. Whilst there are algorithms to aid siRNA design, hairpin-specific characteristics such as stem-length and siRNA core placement within the stem are not well defined. RESULTS: Using more than 91 hairpins designed against HIV-1 Tat and Vpu, we investigated the influence of both of these factors on suppressive activity, and found that stem length does not correspond with predictable changes in suppressive activity. We also detected multiple processed products for all stem lengths tested. However, the entire length of the hairpin stem was not equally processed into active products. As such, the placement of the siRNA core at the base terminus was critical for activity. CONCLUSION: We conclude that there is no fixed correlation between stem length and suppressive activity. Instead, core selection and placement likely have a greater influence on the effectiveness of shRNA-based silencing.

A comparison of multiple shRNA expression methods for combinatorial RNAi.

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.

Multiple shRNA combinations for near-complete coverage of all HIV-1 strains.

BACKGROUND: Combinatorial RNA interference (co-RNAi) approaches are needed to account for viral variability in treating HIV-1 with RNAi, as single short hairpin RNAs (shRNA) are rapidly rendered ineffective by resistant strains. Current work suggests that 4 simultaneously expressed shRNAs may prevent the emergence of resistant strains. RESULTS: In this study we assembled combinations of highly-conserved shRNAs to target as many HIV-1 strains as possible. We analyzed intersecting conservations of 10 shRNAs to find combinations with 4+ matching the maximum number of strains using 1220+ HIV-1 sequences from the Los Alamos National Laboratory (LANL). We built 26 combinations of 2 to 7 shRNAs with up to 87% coverage for all known strains and 100% coverage of clade B subtypes, and characterized their intrinsic suppressive activities in transient expression assays. We found that all combinations had high combined suppressive activities, though there were also large changes in the individual activities of the component shRNAs in our multiple expression cassette configurations. CONCLUSION: By considering the intersecting conservations of shRNA combinations we have shown that it is possible to assemble combinations of 6 and 7 highly active, highly conserved shRNAs such that there is always at least 4 shRNAs within each combination covering all currently known variants of entire HIV-1 subtypes. By extension, it may be possible to combine several combinations for complete global coverage of HIV-1 variants.

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

BACKGROUND: Multiple short hairpin RNA (shRNA) gene therapy strategies are currently being investigated for treating viral diseases such as HIV-1. It is important to use several different shRNAs to prevent the emergence of treatment-resistant strains. However, there is evidence that repeated expression cassettes delivered via lentiviral vectors may be subject to recombination-mediated repeat deletion of 1 or more cassettes. RESULTS: The aim of this study was to determine the frequency of deletion for 2 to 6 repeated shRNA cassettes and mathematically model the outcomes of different frequencies of deletion in gene therapy scenarios. We created 500+ clonal cell lines and found deletion frequencies ranging from 2 to 36% for most combinations. While the central positions were the most frequently deleted, there was no obvious correlation between the frequency or extent of deletion and the number of cassettes per combination. We modeled the progression of infection using combinations of 6 shRNAs with varying degrees of deletion. Our in silico modeling indicated that if at least half of the transduced cells retained 4 or more shRNAs, the percentage of cells harboring multiple-shRNA resistant viral strains could be suppressed to < 0.1% after 13 years. This scenario afforded a similar protection to all transduced cells containing the full complement of 6 shRNAs. CONCLUSION: Deletion of repeated expression cassettes within lentiviral vectors of up to 6 shRNAs can be significant. However, our modeling showed that the deletion frequencies observed here for 6x shRNA combinations was low enough that the in vivo suppression of replication and escape mutants will likely still be effective.

Design and cloning strategies for constructing shRNA expression vectors.

BACKGROUND: Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). RESULTS: We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20-40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression. CONCLUSION: In this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process.