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Manufacture of oligonucleotides
In contrast to the biopharmaceuticals thus far discussed (recombinant proteins and gene therapy plasmids), 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. The basic synthetic strategy is very similar in concept to the means by which peptides are synthesized via the Merrifield method, as described in Chapter 2 (Box 2.1). The nucleotides required (themselves either modified or unmodified, as desired) are first reacted with a protecting chemical group. Each protected nucleotide is then coupled in turn to the growing end of the nucleotide chain, itself attached to a
solid phase. After coupling, the original protecting group is removed and, when chain synthesis is complete, the bond anchoring the chemical to the solid phase is hydrolysed, releasing the free oligo. This may then be purified by HPLC. The most common synthetic method used is known as the phosphoramidite method, which uses a dimethoxytrityl (DMTr) protecting group and tetrazole as the coupling agent. Automated synthesizers are commercially available which can quickly and inexpensively synthesize oligos of over 100 nucleotides.
Vitravene, an approved antisense agent
On 26 August 1998, Vitravene became the first (and thus far apparently the only) antisense product to be approved for general medical use by the FDA. It gained approval within the European Union the following year, although it has since been withdrawn from the EU market for commercial rather than technical reasons. Vitravene is the trade name given to a 21-nucleotide phosphorothioate based product of the following base sequence:
Developed by the US company Isis, Vitravene is used to treat cytomegalovirus (CMV) retinitis in AIDS patients. It is formulated as a sterile solution in WFI (Chapter 3) using a bicarbonate buffer to maintain a final product pH of 8.7. Administration is by direct injection into the eye (intravitreal injection) and each ml of product contains 6.6 mg of active ingredient.
The product inhibits replication of human cytomegalovirus (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.
Antigene sequences and ribozymes
Antigene sequences and ribozymes form two additional classes of antisense agents. However, the therapeutic potential of these agents is only now beginning to be appraised. Certain RNA sequences can function as catalysts. These so-called ‘ribozymes’ function to catalyse cleavage at specific sequences in a specific mRNA substrate. Many ribozymes will cleave their target mRNA where there exists a particular triplet nucleotide sequence G-U-C. Statistically, it is likely that this triplet will occur at least once in most mRNAs.
Ribozymes can be directed to a specific mRNA by introducing short-flanking oligonucleotides, which are complementary to the target mRNA (Figure 11.15). The resultant cleavage of the target obviously prevents translation. One potential advantage of ribozymes is that, as catalytic agents, a single molecule could likely destroy thousands of copies of the target mRNA. Such a drug should, therefore, be very potent.
‘Antigene’ (oligonucleotide) sequences function to inhibit transcription of a specific gene (as opposed to inhibition of translation of a mRNA species). These oligonucleotides achieve this by hybridizing with appropriate stretches of double-stranded DNA, forming a triple helix. This inhibits initiation of transcription of the genes in this region.
The binding of antigene sequences occurs only in the so-called ‘major groove’ of DNA. The incoming oligonucleotide does not disrupt the double-stranded DNA. It binds to it, forming what are termed ‘Hoogsteen base pairs’ —each base in the antigene sequence forming two new
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Nucleotide sequence at which — ribozyme can cleave
-C -C - G
Ñ - G
sequence of the appropriate mRNA via complementary base pairing
Figure 11.15. Outline of how ribozyme technology could prevent translation of specific mRNA, thus preventing synthesis of a specific target protein
hydrogen bonds with a purine base in the targeted region of the double helix. Much research, however, must be undertaken before it will become clear whether such antigene sequences will be of therapeutic use.
Every few decades, a medical innovation is perfected that profoundly influences the practice of medicine. Widespread vaccination against common infectious agents and the discovery of antibiotics serve as two such examples. Many scientists now believe that the potential of gene therapy and antisense technology rivals even the most significant medical advances achieved to date.