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Applegate T.L.,Johnson and Johnson Research Pty Ltd | Applegate T.L.,University of New South Wales | Birkett D.J.,Johnson and Johnson Research Pty Ltd | Mcintyre G.J.,Johnson and Johnson Research Pty Ltd | And 5 more authors.
Retrovirology | Year: 2010

Background: Gene therapy has the potential to counter problems that still hamper standard HIV antiretroviral therapy, such as toxicity, patient adherence and the development of resistance. RNA interference can suppress HIV replication as a gene therapeutic via expressed short hairpin RNAs (shRNAs). It is now clear that multiple shRNAs will likely be required to suppress infection and prevent the emergence of resistant virus.Results: We have developed the first biologically relevant stochastic model in which multiple shRNAs are introduced into CD34+ hematopoietic stem cells. This model has been used to track the production of gene-containing CD4+ T cells, the degree of HIV infection, and the development of HIV resistance in lymphoid tissue for 13 years. In this model, we found that at least four active shRNAs were required to suppress HIV infection/replication effectively and prevent the development of resistance. The inhibition of incoming virus was shown to be critical for effective treatment. The low potential for resistance development that we found is largely due to a pool of replicating wild-type HIV that is maintained in non-gene containing CD4+ T cells. This wild-type HIV effectively out-competes emerging viral strains, maintaining the viral status quo.Conclusions: The presence of a group of cells that lack the gene therapeutic and is available for infection by wild-type virus appears to mitigate the development of resistance observed with systemic antiretroviral therapy. © 2010 Applegate et al; licensee BioMed Central Ltd. Source

Mcintyre G.J.,Johnson and Johnson Research Pty Ltd | Mcintyre G.J.,University of New South Wales | Yu Y.-S.,Johnson and Johnson Research Pty Ltd | Yu Y.-S.,University of New South Wales | And 2 more authors.
BMC Molecular Biology | Year: 2011

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. © 2011 Mcintyre et al; licensee BioMed Central Ltd. Source

Mcintyre G.J.,Johnson and Johnson Research Pty Ltd | Groneman J.L.,Johnson and Johnson Research Pty Ltd | Yu Y.-H.,Johnson and Johnson Research Pty Ltd | Tran A.,Johnson and Johnson Research Pty Ltd | Applegate T.L.,Johnson and Johnson Research Pty Ltd
AIDS Research and Therapy | Year: 2011

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. © 2011 Mcintyre et al; licensee BioMed Central Ltd. Source

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