SiRNA vector

Park J, Nadeau P, Zucah JR, Johnson CM, Mergia A (2005) Inhibition of simian immunodeficiency virus by foamy virus vectors expressing siRNAs. Virology 343 275-282... 

RNAi technology has obvious therapeutic potential as an antisense agent, and initial therapeutic targets of RNAi include viral infection, neurological diseases and cancer therapy. The synthesis of dsRNA displaying the desired nucleotide sequence is straightforward. However, as in the case of additional nucleic-acid-based therapeutic approaches, major technical hurdles remain to be overcome before RNAi becomes a therapeutic reality. Naked unmodified siRNAs for example display a serum half-life of less than 1 min, due to serum nuclease degradation. Approaches to improve the RNAi pharmacokinetic profile include chemical modification of the nucleotide backbone, to render it nuclease resistant, and the use of viral or non-viral vectors, to achieve safe product delivery to cells. As such, the jury remains out in terms of the development and approval of RNAi-based medicines, in the short to medium term at least. 

Amarzguioui, M., Rossi, J.J., and Kim, D. 2005. Approaches for chemically synthesized siRNA and vector-mediated RNAi. FEBS Letters 579(26), 5974-5981. 

Rescue experiments Target noncoding sequence 3 UTRs, alternatively 5 UTRs, can be targeted by siRNA rescue can be performed by vector-encoded target genes lacking UTRs... 

Efficiency of stably integrated shRNA vectors should be confirmed in vitro. Therefore, expand two positives clones per shRNA construct on feeders, freeze aliquots, and cultivate the cells feeder-free on gelatine-coated 10 cm dishes for at least 2 days. Harvest cells and analyze the four clones for knockdown efficiency as described in steps 2 and 3 of Subheading 3.1.2. Determine the best performing clone for each siRNA target sequence. 

PEI grafting on chitosan is becoming a popular approach for modification of chitosan for gene delivery applications. In recent years several efforts to develop chitosan-g-PEI delivery vectors have been published. These include incorporation of mannose [117] and folic acid [118] derivatives for targeted delivery and application of chitosan-g-PEI for the delivery of siRNA [119]. 

Bouxsein NF, McAllister CS, Ewert KK, Samuel CE, Safinya CR (2007) Structure and gene silencing activities of monovalent and pentavalent cationic lipid vectors complexed with siRNA. Biochemistry 46 4785 -792... 

Therefore, improved chemical synthesis strategies for the design of tailored polymeric gene vectors and more information on transfection mechanisms will be needed for the optimization of DNA and siRNA vehicles. 

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