Antisense synthesis

Many human diseases are caused when certain proteins are either over- or underexpressed. Eor example, breast cancer can be induced by overexpressing certain cellular oncogenes within mammary tissue. To study the disease, researchers produce a line of transgenic mice that synthesize an abnormal amount of the same protein. This leads to symptoms of the disease in mice that are similar to what is found in humans. A protein can be overexpressed by inserting a DNA constmct with a strong promotor. Conversely, underexpression of a protein can be achieved by inserting a DNA constmct that makes antisense RNA. This latter blocks protein synthesis because the antisense RNA binds and inactivates the sense mRNA that codes for the protein. Once a line of mice is developed, treatments are studied in mice before these therapies are appHed to humans. 

Bramley, P. et al.. Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5, Plant J. 2 (3), 343, 1992. 

Caruthers, M. H., Synthesis of oligonucleotides and oligonucleotide analogues, in Oligodeoxynucleotides — Antisense Inhibitors of Gene Expression, Cohen, J. S., Ed., CRC Press, Boca Raton, FL, 1989, 7. 

FIG. 65. Solid-phase synthesis of mannosylated dendrons for antisense gene delivery ... 

Mackellar, C., Graham, D., Will, D.W., Burgess, S., and Brown, T., Synthesis and physical properties of anti-HIV antisense oligonucleotides bearing terminal lipophilic groups, Nucleic Acid Research, 1992, 20, 3411-3417. 

Figure 14.14 Outline of how an antisense oligonucleotide can prevent synthesis of a gene product by blocking translation. In practice, antisense oligos are 12-18 nucleotides in length. In many instances, antisense binding is believed to occur in the nucleus...

The product inhibits replication of human CMV (HCMV) via an antisense mechanism. Its nucleotide sequence is complementary to a sequence in mRNA transcripts of the major immediate early region (IE2 region) of HCMV. These mRNAs code for several essential viral proteins and blocking their synthesis effectively inhibits viral replication. 

In contrast to the biopharmaceuticals discussed thus far (recombinant proteins and gene therapy products), antisense oligonucleotides are manufactured by direct chemical synthesis. Organic synthetic pathways have been developed, optimized and commercialized for some time, as oligonucleotides are widely used reagents in molecular biology. They are required as primers, probes and for the purposes of site-directed mutagenesis. 

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. 

Reed MW, et al. Synthesis and evaluation of nuclear targeting peptide-antisense ohgodeoxynucleotide conjugates. Bioconjug Chem 1995 6 101-108. 

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. 

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