Nuclease-based systems

Nuclease-based systems (like the CRISPR/Cas9 system) represent a relatively recent development and a significant improvement over traditional embryonic stem (ES) cell gene... 

Kleimnan et al. 2008). In addition, synthetic siRNAs are also subject to degradation in vivo by nuclease activity. Besides side effects and instability, the efficient and specific delivery of the RNAi indncers to the target cell still requires optimization. Here we snmmarize the cnrrent statns of nncleic acid-based antiviral therapentics. The focns will be on antiviral strategies nsing antisense and RNAi technology. Additionally, antiviral ribozymes and aptamers will be discussed briefly, with a focus on recent studies. Gene therapy approaches and delivery systems are the subject of Chapter 11 of this book. 

Of the various methods that may be used to determine bioavailability for ASOs, the best refer to tissue levels as the most relevant metric for calculating an estimate of absolute bioavailability. As mentioned in Chapter 4, ASOs distribute rapidly to the tissues, with an extremely slow transfer rate back into the central circulation. In addition, the elimination of ASOs occurs predominantly by nucleases in the tissue compartment. Thus, bioavailability based on plasma concentrations does not provide an accurate estimate of absolute bioavailability for ASOs if plasma concentrations cannot be quantified at extremely low concentrations for a prolonged period of time to adequately assess systemic exposure. The direct use of tissue levels in combination with physiologic pharmacokinetic modeling, however, may allow the accurate determination of bioavailability for ASOs. 

Another important set of multiplexed assays monitor mRNA transcript levels. The expression level of all the genes involved in a known signal transduction pathway or other selective genes can be monitored simultaneously as a way of following compound effects on a cell. The current technologies for multiple mRNA detection include quantitative reverse transcriptional PCR (qRT-PCR), qNPA (quantitative nuclease protection assays), mass array assay technologies and branched DNA detection on Luminex beads (Panomics). The applications of such multiplexed in vitro and cell-based detection systems should provide more predicative information in hit finding and lead characterisation. 

In regard with NER, at least 32 proteins, forming several complexes, are necessary and sufficient in a successful repair of a bulky lesion. In the case of BER, 5-6 proteins are necessary to reconstitute the entire process on a single base damage [7]. For both systems, the action ofthe proteins or complexes is coordinated and protein interactions allow passing from one step to the next one. Thus, free repair intermediates which could be entry sites for nucleases that would degrade DNA, are avoided. 

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