RNA interference (RNAi) refers to the process by which dsRNA molecules silence a target through the specific destruction of their mRNA molecules. Subsequent to the discovery that small interfering RNAs (siRNAs) mimicking the Dicer cleavage products can silence mammalian genes, RNAi has become the experimental tool of choice to suppress gene expression in a wide variety of organisms. In addition, RNAi has also become a method of choice for key steps in the development of therapeutic agents, from target discovery and validation to the analysis of the mechanisms of action of small molecules. To date, several strategies have been devised to trigger the RNAi pathway, each of which is adapted and optimized for different cell systems. Although the technology has several advantages over other methods, the specificity of gene silencing is not absolute and there is a danger of off-target effects and activation of innate immunity. Also, strategic success of therapeutic siRNAs will depend on the development of a delivery vehicle that can target pathogenic cells and from our understanding of the biogenesis of microRNAs (miRNAs). The purpose of this book is to provide the readers with the recent advances in siRNA design, expression, delivery, in vivo imaging, and methods to minimize siRNA unwanted effects and use in patients.
To design an effective siRNA, one must consider the base composition of the chosen site and whether the target site will be accessible. Chapter 1 critically reviews the published design guide rules and presents new statistical and clustering design strategies that are useful for selecting effective siRNA sequences. If the chosen target is an RNA virus that can mutate rapidly, one may consider to target conserved site sequences and/or to combine diverse siRNA sequences.
New books on Target Discovery and Validation Reviews and Protocols
Volume 1 focuses on novel and innovative techniques, and presents the most up-to-date protocols available for maximizing the likelihood of achieving target-selective inhibition in vivo while minimizing side effects. The profound impact of genomics, proteomics and bioinformatics on target discovery is explored, and specific attention is given to the role of transgenic and knockout animals in functional genomics and target validation. Cancer researchers will find tremendous value in the molecular classification of breast cancers and the review of protocols for tumor antigens and cancer vaccines. The methods and protocols collected here, all reviewed by leading scientists and clinicians, present the practical details necessary for translating the enormous discovery potential of the genome into real therapeutic products.
Volume 2 collects all the practical details required for efficient translation of discovered targets into real pharmaceutical drugs. Specific targets in cancers and autoimmunity are described and the potential of using siRNAs, antisense oligonucleotides and RNA aptamers in patients is reviewed. This volume explores the tremendous impact of the application of genotyping and gene expression profiling on the future of healthcare, and presents cutting-edge protocols to aid in bringing agents against specific targets closer to application in the clinic. Collectively, these volumes provide a thorough review of the most cutting-edge methods available for each step in drug target identification, validation, and clinical application. For researchers, an understanding of available methods aids in the creation of innovative experiments in the laboratory, and the successful translation of target discovery to real therapeutics.
Small interfering RNAs activate innate immunity via endosomal Toll-like receptor 8
In the present paper published in the Journal of Molecular Biology, I have found that the immunostimulatory effects of siRNA were sequence-dependent, since only certain sequences are prone to induce inflammatory responses while other not. Interestingly, the induction inflammatory cytokines such TNF-a, IL-6 and interferon INF-a in adherent PBMC are not restricted to double-stranded siRNA, because induction was also obtained with single-stranded siRNAs (sense or antisense strands). Immunostimulation requires the endosomal localization of siRNAs, since it was chloroquine-sensitive and was not seen when siRNAs were delivered directly to the cytoplasm via electroporation.
The data indicate for the first time that the activation of the innate immunity genes by siRNAs in adherent freshly isolated PBMC and blood monocytes is mediated via endosomal Toll-like receptor 8. This novel finding would indicate that endocytosed double-stranded siRNAs are denatured in the endosomal and/or endolysosomal compartments to generate single-stranded sense and antisense strands, which trigger Toll-like receptor 8 (and perhaps TLR 7) as illustrated bellow. Although CU nucleotides seem to enhance the immunoactivity of siRNAs, other sequences are also involved.
Induction of Inflammatory Cytokines and Interferon Responses by Double-stranded and Single-stranded siRNAs is Sequence-dependent and Requires Endosomal Localization
Download article in PDF format
Also available online at www.sciencedirect.com
In this completely updated book experts take advantage of the latest advances in ribozyme, DNAsyme, and RNA interference technology to describe in details the exciting and successful methods now available for gene inactivation in vitro and in vivo. Each proven protocol includes a background introduction outlining the principle behind the technique, step-by-step instructions and tips on troubleshooting and avoiding known pitfalls. The methods may be used to analyse gene function and target validation, and to develop new therapeutics for human diseases.
Naturally occurring antisense RNAs can make sense
The expression of antisense RNAs has been explored as an experimental and therapeutic tool. However, the extend to which naturally expressed antisense RNAs occur in mammalian cells has not been addressed experimentally. In this January issue of Nature Biotechnology [vol 22 (1)], we described the first experimental strategy for the systematic analysis of sense-antisense transcripts in mammalian cells. Notably, the identification of a large number of antisense RNAs suggests that regulation of gene expression by double-stranded RNA could be a general mechanism in mammalian cells. Interestingly, several identified antisense RNAs were found to be complementary to their spliced mRNAs over several exons, suggesting that they are generated via the activity RNA-dependent RNA polymerase.