Antisense modifications

Modification of the Phosphodiester Backbone. Oligonucleotides having modified phosphate backbones have been extensively studied (46). Because altering the backbone makes derivatives generally more resistant to degradation by cellular nucleases, these materials have the potential to be more resilient antisense dmgs. 

ASON are sequences of usually 17-30 bases of single-stranded DNA that hybridize to specific genes or their mRNA products by Watson-Crick base pairing and disrupt their function. In the case of AS-ODN (antisense oligodeoxyribonucleotides) cellular RNAseH is able to bind to the DNA-RNA duplex and hydrolyze the RNA, resulting in increased transcript turnover. Modifications to the deoxy moiety at the 2 -sugar position prohibits RNAse H action. 

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. 

Aptamers appear to display low immunogenicity but, when administered systemically, they are quickly excreted via size-mediated renal clearance. In order to prevent renal removal, such aptamers are usually conjugated to PEG. PEG may also help further protect the aptamers from degradation by serum nucleases native aptamers are prone to nuclease attack, but their half-lives can most effectively be extended via chemical modification, as discussed earlier in the context of antisense agents. 

Fig. 9.20 Luminescent carbon nanotubes (a) Cy3 labeled antisense myc-modified SWCNTs (b) quantum dots-modifed MWCNTs (With permission from American Scientific publisher and...

Antisense Approach This is a relatively new approach and it requires modifications to oligonucleotides that can bind to RNA and DNA (refer to Appendix 2 for a description of cell structure, genes, DNA, RNA, and proteins). The antisense drugs are used to stop transcriptional (from DNA) or translational (from RNA) pathways from proceeding, and so interfere with the process of disease. 

Example 56 the Isis Pharmaceutical group in their extensive investigations of antisense oligonucleotides as therapeutics has described the synthesis of 3 -C-methylene nucleoside phosphonoamidites for the new backbone modification of oligonucleotides [90]. This paper gives good insight into tricoordinate phosphorus and related H-phosphonate chemistry in the service of nucleotide synthesis. 

A number of different chemical modifications of the basic RNA antisense have shown promise in the lab and in animal test systems,... 

Kurreck, J. Antisense Technologies Improvement through Novel Chemical Modifications. European Journal of Biochemistry 270 no. 8 (2003) 1628-1644. 

Genetic Modification of Flavonoid Production Using Inhibition of Flavonoid Biosynthetic Gene Activity by Sense or Antisense RNA... 

Colliver, S.P., Morris, P., and Robbins, M.P., Differential modification of flavonoid and isofiavonoid biosynthesis with an antisense chalcone synthase construct in transgenic Lotus corniculatus. Plant Mol. Biol, 35, 509, 1997. 

Carron, T.R., Robbins, M.P., and Morris, P., Genetic modification of condensed tannin biosynthesis in Lotus corniculatus. 1. Heterologous antisense dihydroflavonol reductase down-regulates tannin accumulation in hairy root cultures. Theor. Appl. Genet., 87, 153, 1994. 

Zuker, A. et al.. Modification of flower colour and fragrance by antisense suppression of the flavanone 3-hydroxylase gene. Mol Breed., 9, 33, 2002. 

Several other types of backbone modification have also been proposed, which produce nuclease-resistant oligos. Of these, a-oligos have been extensively studied. In a-oligos the base is transposed from the natural P-orientation to the unnatural a-orientation to form a parallel duplex with target sequence. This parallel duplex is nuclease-resistant, but does not elicit RNase H activity (Cazenave et al., 1989). These modifications have generated limited interest and application in antisense research. 

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. 

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