Target Discovery and Validation Reviews and Protocols

8 Sioud
serum biomarkers. This synergism is also true for the analysis of gene biomarkers
detected by DNA microarrys. Given the importance of serological biomarkers, we
have developed a protein microarray containing several immunoselected phagedisplayed
peptides and shown its application in breast cancer diagnosis (28).
Chapter 17, Volume 1, describes this novel approach and highlights the importance
of protein arrays in target discovery and validation (16).
1.2. Target Validation
Once a gene target or a mechanistic pathway is identified, the next step is
to demonstrate that it does play a critical role in disease initiation, perpetuation,
or both. A range of in vitro assays exists for modulating gene expression in vitro
and in vivo. These include the use of antibodies, dominant negative controls,
antisense oligonucleotides, locked nucleic acids, peptide nucleic acids, ribozymes,
and siRNAs. There are several notable examples in which these technologies
have been targeted against genes with already well-characterized roles in disease
states and these examples have served as a proof of concept for their use
in target validation (16).
Today, the RNAi technology seems to offer a number of significant advantages
over other strategies, including increased specificity (16). Although prediction
algorithms have begun to offer some guidance for siRNA sequence selection,
identification of highly effective and gene-specific siRNA and their delivery
to human cells is often problematic, and various factors may influence siRNA
action. Chapter 9, Volume 2, describes the latest guidelines for designing effective
siRNAs for functional genomics and target validation studies. Also, the
described rules should potentially enhance our ability to design more effective
hairpin siRNA libraries. By using a cell array, target discovery and validation using
siRNAs can be performed in a high-throughput format (see Chapter 9, Volume 1).
The great advantage of protein knockdown through mRNA modulation is that only
sequence information about the target gene is required. A critical issue for the use
of synthetic siRNAs in target validation is the choice of controls and siRNA
sequences. One should therefore verify that the sequence-dependent off-target
effects are low and that the designed siRNAs are not immunostimulatory (29).
Genetically modified animals that either overexpress (transgenic animals) or
no longer express the target (knockout animals) are extremely useful in the target
validation and drug discovery processes. These models represent the best available
tool for examining the complex interaction that underlines pathological
responses. When the target is a single gene, target validation will frequently
involve producing a transgenic animal that carries either the wild-type gene or its
mutated form to demonstrate that this animal is either overexpressing the wildtype
gene or that the mutated gene has a phenotype that mimics a certain aspect
of the human disease. Subsequently, drug screening can be performed in this animal
to show that modulation of transgene expression can have therapeutic effects.