A direct comparison of strategies for combinatorial RNA interference

<p>Abstract</p> <p>Background</p> <p>Combinatorial RNA interference (co-RNAi) is a valuable tool for highly effective gene suppression of single and multiple-genes targets, and can be used to prevent the escape of mutation-prone transcripts. There are currently three main approaches used to achieve co-RNAi in animal cells; multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and miRNA-embedded shRNAs, however, the relative effectiveness of each is not known. The current study directly compares the ability of each co-RNAi method to deliver pre-validated siRNA molecules to the same gene targets.</p> <p>Results</p> <p>Double-shRNA expression vectors were generated for each co-RNAi platform and their ability to suppress both single and double-gene reporter targets were compared. The most reliable and effective gene silencing was achieved from the multiple promoter/shRNA approach, as this method induced additive suppression of single-gene targets and equally effective knockdown of double-gene targets. Although both lhRNA and microRNA-embedded strategies provided efficient gene knockdown, suppression levels were inconsistent and activity varied greatly for different siRNAs tested. Furthermore, it appeared that not only the position of siRNAs within these multi-shRNA constructs impacted upon silencing activity, but also local properties of each individual molecule. In addition, it was also found that the insertion of up to five promoter/shRNA cassettes into a single construct did not negatively affect the efficacy of each individual shRNA.</p> <p>Conclusions</p> <p>By directly comparing the ability of shRNAs delivered from different co-RNA platforms to initiate knockdown of the same gene targets, we found that multiple U6/shRNA cassettes offered the most reliable and predictable suppression of both single and multiple-gene targets. These results highlight some important strengths and pitfalls of the currently used methods for multiple shRNA delivery, and provide valuable insights for the design and application of reliable co-RNAi.</p

Computational Models of HIV-1 Resistance to Gene Therapy Elucidate Therapy Design Principles

Gene therapy is an emerging alternative to conventional anti-HIV-1 drugs, and can potentially control the virus while alleviating major limitations of current approaches. Yet, HIV-1's ability to rapidly acquire mutations and escape therapy presents a critical challenge to any novel treatment paradigm. Viral escape is thus a key consideration in the design of any gene-based technique. We develop a computational model of HIV's evolutionary dynamics in vivo in the presence of a genetic therapy to explore the impact of therapy parameters and strategies on the development of resistance. Our model is generic and captures the properties of a broad class of gene-based agents that inhibit early stages of the viral life cycle. We highlight the differences in viral resistance dynamics between gene and standard antiretroviral therapies, and identify key factors that impact long-term viral suppression. In particular, we underscore the importance of mutationally-induced viral fitness losses in cells that are not genetically modified, as these can severely constrain the replication of resistant virus. We also propose and investigate a novel treatment strategy that leverages upon gene therapy's unique capacity to deliver different genes to distinct cell populations, and we find that such a strategy can dramatically improve efficacy when used judiciously within a certain parametric regime. Finally, we revisit a previously-suggested idea of improving clinical outcomes by boosting the proliferation of the genetically-modified cells, but we find that such an approach has mixed effects on resistance dynamics. Our results provide insights into the short- and long-term effects of gene therapy and the role of its key properties in the evolution of resistance, which can serve as guidelines for the choice and optimization of effective therapeutic agents

RNA-mediated inhibition of HIV in a gene therapy setting

At present, treatment for HIV-1 infection employs highly active anti-retroviral therapy (HAART), which utilizes a combination of RT and protease inhibitors. Unfortunately, HIV can escape many therapies because of its high mutation rate and the complexity of its pathogenesis. HIV-1 integrates into the cellular genome, which facilitates persistence and acts as a reservoir for reactivation and replication. As an alternative or adjuvant to chemotherapy we have been developing an RNA-based gene therapy approach for the treatment of HIV-1 infection. This article summarizes the various RNA based technologies that we have developed for potential application in a gene therapy setting

RNA-mediated inhibition of HIV in a gene therapy setting

At present, treatment for HIV-1 infection employs highly active anti-retroviral therapy (HAART), which utilizes a combination of RT and protease inhibitors. Unfortunately, HIV can escape many therapies because of its high mutation rate and the complexity of its pathogenesis. HIV-1 integrates into the cellular genome, which facilitates persistence and acts as a reservoir for reactivation and replication. As an alternative or adjuvant to chemotherapy we have been developing an RNA-based gene therapy approach for the treatment of HIV-1 infection. This article summarizes the various RNA based technologies that we have developed for potential application in a gene therapy setting

Intracellular ribozyme applications

The exquisite target selectivity of trans -acting ribozymes has fostered their use as potential therapeutic agents and tools for down-regulating cellular transcripts. In living cells, free diffusion of RNAs is extremely limited, if it exists at all. Thus, getting ribozymes to base-pair with their cognate targets requires co-localizing the ribozyme transcript with the target RNA. In addition, not all sites along a given target RNA are equally accessible to ribozyme base pairing. Cellular proteins greatly influence the trafficking and structure of RNA, and therefore making ribozymes work effectively in cells a significant challenge. This article addresses the problems of getting engineered ribozymes to effectively pair with and cleave targets in cells. The work described here illuminates methods for target-site selection on native mRNAs, methods for ribozyme expression, and strategies for obtaining a discrete intracellular localization of ribozymes

Intracellular ribozyme applications

The exquisite target selectivity of trans -acting ribozymes has fostered their use as potential therapeutic agents and tools for down-regulating cellular transcripts. In living cells, free diffusion of RNAs is extremely limited, if it exists at all. Thus, getting ribozymes to base-pair with their cognate targets requires co-localizing the ribozyme transcript with the target RNA. In addition, not all sites along a given target RNA are equally accessible to ribozyme base pairing. Cellular proteins greatly influence the trafficking and structure of RNA, and therefore making ribozymes work effectively in cells a significant challenge. This article addresses the problems of getting engineered ribozymes to effectively pair with and cleave targets in cells. The work described here illuminates methods for target-site selection on native mRNAs, methods for ribozyme expression, and strategies for obtaining a discrete intracellular localization of ribozymes

Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs

A primary advantage of lentiviral vectors is their ability to pass through the nuclear envelope into the cell nucleus thereby allowing transduction of nondividing cells. Using HIV-based lentiviral vectors, we delivered an anti-CCR5 ribozyme (CCR5RZ), a nucleolar localizing TAR RNA decoy, or Pol III-expressed siRNA genes into cultured and primary cells. The CCR5RZ is driven by the adenoviral VA1 Pol III promoter, while the human U6 snRNA Pol III-transcribed TAR decoy is embedded in a U16 snoRNA (designated U16TAR), and the siRNAs were expressed from the human U6 Pol III promoter. The transduction efficiencies of these vectors ranged from 96-98% in 293 cells to 15-20% in primary PBMCs. A combination of the CCR5RZ and U16TAR decoy in a single vector backbone gave enhanced protection against HIV-1 challenge in a selective survival assay in both primary T cells and CD34(+)-derived monocytes. The lentiviral vector backbone-expressed siRNAs also showed potent inhibition of p24 expression in PBMCs challenged with HIV-1. Overall our results demonstrate that the lentiviral-based vectors can efficiently deliver single constructs as well as combinations of Pol III therapeutic expression units into primary hematopoietic cells for anti-HIV gene therapy and hold promise for stem or T-cell-based gene therapy for HIV-1 infection