Target validation through protein- domain knockout – applications of intracellularly stable single-chain antibodies

1477-3627/02/$ – see front matter ©2002 Elsevier Science Ltd.All rights reserved. PII: S1477-3627(01)02172-9 Sequencing of the human genome has provided the pharmaceutical industry with thousands of new potential drug targets, most of which operate intracellularly [1,2]. This fact opens new perspectives for therapy of human diseases but also demands new approaches in target validation. Different experimental tools are currently used for validation of these emerging intracellular drug targets. The classical way to investigate the function and thereby determine the physiological and pathological relevance of gene products is to interfere with the expression of the gene. Gene knockout in mice by performing homologous recombination in mouse embryonic stem cells (ES cells) is a commonly applied approach for target validation, acting at the genomic level. The strength of this technology is its power for investigation of the function of a gene in the whole mammalian organism. Nevertheless, the achievement of a gene knockout is time-consuming, laborious and not applicable in human cells. In addition, compensatory effects or, conversely, complex phenotypes caused by the lack of varied protein functions frequently impair the dissection of specific functions of a gene product (for a review see Ref. [3]). Hence, from the phenotype of a knockout mouse, one often cannot extrapolate the effects of a putative drug action, as ‘smart’ modern drugs usually influence specific functions of protein domains (e.g. by binding to the ATP-binding cassette of a kinase domain, as exemplified by Gleevec) rather than eliminate all interactions of the target protein with the proteome. Antisense and RNA interference (RNAi) approaches intend to perform mRNA knockdowns. In principle, the idea behind an antisense approach is very simple: Base pairing of the antisense RNA (or DNA) with its corresponding mRNA is thought to block translation of the mRNA either by preventing the access of the protein translation machinery or by marking the RNA for degradation by RNase H [4,5]. An advantage of this technology is the possibility of rapid and cost-effective generation of specific antisense oligonucleotides. Nevertheless, antisense technology has to overcome several obstacles such as low stability, toxicity, identification of accessible mRNA sequences, specificity problems and the generation of immune responses (for a comprehensive review see Refs [5,6]). Today, some but not all of these problems have been solved by chemical modifications of the antisense RNA backbones [5]. Yet, Target validation through proteindomain knockout – applications of intracellularly stable single-chain antibodies Peter Lichtlen, Adrian Auf der Maur and Alcide Barberis