Antisense Technology As A Tool For Gene Functional1zation And Target Validation

The sequencing of the human and other genomes has resulted in intense interest in the development of tools with which to determine the roles of various gene products and to de termine whether they are appropriate targets for drug therapy. With advances in automation, antisense technology has proved to be a versatile, effective tool for these purposes (158). The advances in automation include rapid small-scale synthesizers that can synthesize antisense inhibitors in a 96-well-plate format. These are coupled to in-line automated analytical methods that provide quality assurance. The antisense inhibitors are then screened in vitro by the use of an automated reverse transcriptase/polynuclease chain reaction (RT-PCR).With these advances, it is now possible to create antisense inhibitors to hundreds to thousands of genes per year. Figure 5.2 provides a general scheme for rapid creation and evaluation of antisense inhibitors. Figure 5.3 shows a typical 96-well-plate screen to determine the optimal site for antisense effects, in this case for TNFa-receptor 1. In this assay, antisense inhibitors to 80 sites in the target RNA are synthesized and screened in TNFa-receptor 1 positive cells. In addition, the effects of a variety of control oli-gonucleotides are evaluated. In this figure, the RNA is displayed 5' in the translated region to the 3'-untranslated region and each bar rep resents the level of the target RNA, determined by RT-PCR, clearly interacting in several sites and resulting in significant reductions in the target RNA. Based on these data, several antisense inhibitors can be selected for further study, including in vivo evaluation in various animal models.

When one considers the desired attributes for tools for gene functionalization and target validation, it is readily apparent that antisense technology meets these criteria. Antisense inhibitors are gene specific. In the past decade, at Isis Pharmaceuticals alone we have created antisense inhibitors to nearly 1000 genes, so we are confident that the technology can be used for any gene. Having automated antisense, it is rapid, efficient, and cost effective. Antisense inhibitors are versatile in that they can be used for both in vitro and in vivo studies. Antisense inhibitors are pharmacological agents, so they provide direct insights into the types of responses to be expected from acute interventions with drugs that affect the target. Antisense inhibitors can also be used to dissect the roles of splice variants and to identify novel gene functions.

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