Antisense effects

The therapeutic utility of systemically administered ASON had been limited by their short plasma half life (sometimes even less than 3 min). This is due to their sensitivity to nuclease digestion. When the first-generation ASON were chemically modified, e.g., by replacing the oxygen in the phosphodiester bond with sulfur (phosphorothiorate) they obtained an increased stability in biological fluids while their antisense effect has been maintained. First-generation agents can be delivered via intravitreal injection, parenterally, by topical cream, enema, and inhaled aerosol. These antisense... 

A few ex vivo and in vivo studies have been published claiming an antigene (and antisense) effect of mixed purine/pyrimidine sequence PNA [48, 49, 78-80]. However, as pointed out by us in recent reviews [81, 82] these studies lack fundamental controls such as the inclusion of relevant internal standards as a control for sequence-specific non-antigene/antisense effects, thus confirmatory studies are warranted. The in vivo antigene studies from Richelsoris group [79, 83] completely lack a rational basis for the claimed effects. First of all there is no evidence that... 

Good L., Awasthi S.K., Dryselius R., Larsson O., Nielsen P.E. Bactericidal antisense effects of peptide-PNA conjugates. Nature Biotechnol. 2001 19 360-364. 

RNAi and ribozymes represent two additional approaches to gene silencing/down-regulation with therapeutic potential. RNAi is an innate cellular process that achieves silencing of selected genes via an anti-sense mechanism. It shares many characteristics with the antisense-based approach described above, but also some important differences, e.g. in the exact mechanism by which the antisense effect is achieved. 

A prerequisite to acquire an antisense effect is the maintenance of AS-ODN within the target cells. Several studies have reported that the majority of phosphorothioated AS-ODNs taken up by the kidney remains intact for several hours [110,113]. In fact, 4 days after administration, 3% of the infused dose was still present in the kidney intactly [110]. Although several studies have confirmed the presence of intact AS-ODN in the kidney, concomitant metabolism in the kidney of 20% after 6 h [113], 50% after 48 h [118,126] and 50% after 4 days [114] has also been reported. 

It is important to realize however that these antisense molecules were not specifically targeted to the endothehum. Consequently, the contribution of the endothelial cells to the effects observed is unknown. Furthermore, in these studies adequate control experiments with mismatched ohgonucleotides are essential, since polyanionic agents such as antisense oligonucleotides can exert a broad range of non-specific antisense effects due to non-specific binding to proteins [117]. 

To enhance the cellular uptake and nuclease resistance of oligonucleotides, different terminal modifications at the 5 or 3 terminus of oligonucleotides have been attempted. Polylysine, avidin (such as acridine), and cholesterol have been used to improve cellular uptake and antisense effects of oligos (Nechers, 1989, 1993). However, the value of these approaches remains uncertain and needs to be further determined, especially in in vivo settings. 

III. In vitro biological activity specificity and efficiency Cell culture Demonstrate specific antisense effect by various bioassays such as anti-viral and anti-proliferation effect, induction of apoptosis... 

Mizu M, Koumoto K, Anada T et al (2004) Antisense oligonucleotides bound in the polysaccharide complex and the enhanced antisense effect due to the low hydrolysis. Biomaterials 25 3117-3123... 

Kole, R., and Sazani, P. (2001) Antisense effects in the cell nucleus modification of splicing. Curr OpinMol Ther. 3,229-234. 

The scope of the toxicity studies when surrogate molecules have been used includes pharmacology studies, subchronic and chronic studies, reproductive toxicity studies, immunotoxicity studies, and even carcinogenicity studies. Short-term assays like safety pharmacology studies are too short for there to be an antisense effect, so we have not used the surrogate approach in these assays. However, when safety pharmacology endpoints are included in subchronic or chronic studies, surrogates are assessed. 

Sazani P, Kang SH, Maier MA, Wei C, Dillman J, Summerton J, Manoharan M, Kole R. Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs. Nucleic Acids Res. 2001 29 3965-3974. 

Binding and Effects of Binding to Non-nucleic Acid Targets. Phosphorothioate oligonucleotides tend to bind to many proteins and those interactions are influenced by many factors. The effects of binding can influence cell uptake, distribution, metabolism, and excretion. They may induce non-antisense effects that can be mistakenly interpreted as... 

Winkler J, Saadat K, Gavilan MD, Urban E, Noe CR (2009) Oligonucleotide-polyamine conjugates influence of length and position of 2 -attached polyamines rat duplex stability and antisense effect. Eur J Med Chem 44 670-677... 

NADs as therapies for HIV/AIDS. Although inhibition of HIV replication did not result solely from antisense effects, the reports certainly impressed researchers. Because of their superior chemical, biochemical, and biological advantages, OPTs have been studied to a greater extent than other NADs and have more frequently found their way into clinical trials. OPTs, like OMPs, are mixtures of stereoisomers that can adversely affect antisense activity. It was later determined that the stereo-controlled synthesis of OPTs might produce more efficacious antisense ODNs. Dr. Wojciech Stec of the Polish Academy of Science developed the first method for stereospecific synthesis of OPTs and reported that their biological characteristic varies depending on which stereoisomers is used. Unfortunately, all clinical trials have utilized mixtures of stereoisomers. 

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