Target Discovery and Validation Reviews and Protocols

Target Discovery and Validation 5
abolishes expression give advantage to tumor cells. The challenge ahead is to
efficiently translate these epigenetic changes into real therapies. Chapter 2,
Volume 2, highlights the epigenetic alterations in cancers and suggests potential
therapeutic strategies.
When a gene variant is associated with drug response, there is often an obvious
direction to pursue for making clinical use of the polymorphism (12). However,
in complex genetic diseases, risk factors often do not lead to obvious treatment
implications. For example, the discovery of Brca1 and Brca2 genes involved in
hereditary breast cancer has provided critical insight into the mechanisms of
carcinogenesis; however, neither gene is a viable therapeutic target. It is worth
noting that mutations in a single gene can cause severe disease as exemplified
by rare genetic diseases such as cystic fibrosis. With respect to treatment of
these genetic disorders, where the gene responsible for the disease may have
exerted its effect during the developmental process, gene therapy will play a
crucial role.
1.1.2. Forward and Reverse Genetics
The major advantage of using genetic approaches instead of expression profiling
genomics is that there is a clear link between a genetic alteration and a disease
phenotype. Among the commonly used model organisms, the mouse is the
preferred choice because, genetically, it is relatively close to human. Today, most
human diseases can be mimicked in mouse (13). To date forward genetics has
been most extensively performed using the chemical mutagen N-ethylnitrosouera
(ENU) to produce random mutations in the genome (14,15). ENU induces single
base-pair DNA alteration distributed through the genome. In addition to chemical
mutagenesis, random integration of retroviruses or transposons could be the
most desirable screen because the vector sequence provides a molecular fingerprint
for the mutated gene, thereby facilitating its identification.
Although the observed alterations are limited to cellular effects, phenotype
screening in mammalian cells can represent a valuable first method for target
discovery. Loss-of-function genetic screens by using RNA interference (RNAi)
now represents one of the most used technologies (16). RNAi is a powerful natural
cellular process in which double-stranded RNAs (dsRNAs) target homologous
mRNA transcripts for degradation (17). In this process, the dsRNA is
recognized by an RNase III nuclease, which processes the dsRNA into smallinterfering
RNAs (siRNAs) of 19 nucleotides, with two nucleotide 3′ overhangs
(18). Subsequently, these intermediates are incorporated into an RNA-interfering
silencing complex, which contains the proteins needed to unwind the doublestranded
siRNA and cleave the target mRNA at the site where the antisense
RNA is bound. In addition to chemical synthesis, siRNAs can be expressed with
a hairpin loop from plasmids and viral vectors, which offers the opportunity for