A. Principles of Ribozyme-Based Gene Target Validation
The gene inactivation ability of ribozymes has been exploited for pharmaceutical drug target validation. A typical drug target is a gene or gene product (protein) whose inhibition results in a therapeutic effect. However, prior to the expensive process of drug discovery and development, putative drug target genes are often screened or validated by functional genetics approaches. If manipulation of gene expression leads to a desired therapeutic phenotype, then the gene is validated and may be then selected as a candidate drug target. Traditional approaches for revealing gene function include transgene and gene knockout experiments. In transgene experiments, the candidate gene is overexpressed in cells or in animals to increase the amount and activity of the gene product. This "gain-of-function" can lead to an acquired phenotype, which will help to elucidate function of the gene. A limitation to this approach is that the artificially created transgene may not be subject to the native regulation and thus produce an abnormal physiology. Also, the gain-of-function does not reflect the typical antagonistic actions of drugs.
Somatic cell gene knockout procedures, in contrast, produce a "loss-of-function" phenotype that more accurately mimics drug action. The pheno-type that results from a gene knockout can reveal gene function. However, the procedure is usually achieved via DNA recombination, which is complex and not always successful. In addition, many animal knockouts are developmentally lethal, yielding no clues about gene function. Furthermore, the complete and permanent removal of a gene in a gene knockout procedure is not an ideal mimic of the activity of drugs, which only partially and temporarily inhibit their targets. Ribozymes, by contrast, usually only partially inhibit target gene expression and thus better resemble the partial inhibitory activity of drugs, thereby providing an alternative and perhaps more accurate method for target gene validation.
The typical steps for target validation using ribozymes include: (1) determining or developing the functional cell-based assays for disease phenotype, (2) designing ribozymes against the intended target and determining whether the designed ribozymes target only the gene of interest through database searches, (3) construct and prepare ribozyme vectors, (4) transduce cells and perform the functional phenotypic assay for gene validation, and (5) biochemical analysis for mRNA and protein levels in the ribozyme-treated cells to correlate knockdown of the gene to the phenotype alteration. If an expected and desirable phenotype is correlated with gene knockdown, then the gene target is considered functionally validated.
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