Target Validation

A crucial step to validate the most promising molecular targets for drug development is to determine their biological relevance for a given disease. The possible strategies that are usually used to understand the function of a specific gene in a cell are: (1) either based on techniques that impair the expression of the candidate target gene, or alternatively (2) rely on the use of products that act by specifically interfering or inhibiting the function, but not the expression, of the final product. In both cases, the resulting phenotype turns out as a powerful source of information on the function of the target protein.

The generation of null mutants by homologous recombination of a given gene in a single cell or in an entire organism has been extensively used to create models of several human diseases, including cancer. Using this technique (known as gene knockout), in which the gene of interest is irreversibly disrupted and the synthesis of the encoded products abolished, allowed to make an incredible and rapid progress in our understanding of the function of several oncogenes and tumor suppressor genes. As an alternative strategy for highly specific gene silencing the use of RNA interference has proven to be a precious approach that permits loss-of-function phenotypic screens in mammalian somatic cells or in whole animals (see Chapters 9, 10, and 11). Indeed, it is now feasible to design RNAi constructs against virtually any transcript in the genome. Furthermore, in contrast to the knockout approach, the RNA interference-based RNA strategies achieve loss-of-function phenotypes without the loss of genomic information of the targeted gene (recently reviewed in ref. 16).

Fig. 1. Schematic representation of the systematic evolution of ligands by exponential enrichment (SELEX) process. The single-stranded (ss) DNA library is amplified by PCR to generate the double-stranded DNA pool that will be transcribed by T7 RNA polymerase. The pool of RNA molecules with different conformations will be used for the selection process (see Subheading X for details).

Fig. 1. Schematic representation of the systematic evolution of ligands by exponential enrichment (SELEX) process. The single-stranded (ss) DNA library is amplified by PCR to generate the double-stranded DNA pool that will be transcribed by T7 RNA polymerase. The pool of RNA molecules with different conformations will be used for the selection process (see Subheading X for details).

This leaves the possibility to restore the exact expression of the endogenous gene once the RNAi vector is silenced or removed.

However, a major disadvantage of the gene silencing approaches to obtain functional data on a given protein is that, in most cases, proteins and enzymes involved in crucial functions, such as cell growth and differentiation, act in concert with various protein partners thus forming large stable complexes. Therefore, depleting a single key protein from the cell will change, or even disrupt, one or more of these multiprotein complexes recruited by the target protein. As a consequence, the resulting phenotype will be produced by the simultaneous impairment of several protein functions and the understanding frequently ambiguous. Furthermore, silencing a gene gives no information about which part or domain of the protein is important for its function. Therefore, to overcome these problems, additional approaches have been favored that enable to interfere with a given protein function without interfering with its expression. Indeed, addressing the process of target validation using inhibitors of protein function that directly target the protein in a drug-like manner has the advantage to interfere with a protein activity with low destabiliza-tion of the proteomic status of the cell. As excellent alternatives to monoclonal antibodies, peptides and small-molecule inhibitors, RNA-based aptamers have recently proven to be efficient inhibitors of a wide variety of protein implicated in cancer. Indeed, aptamers can recognize within the same protein different domains, or posttranslational modifications, which allows functional analysis of proteins at the molecular level. Furthermore, the SELEX procedure permits to generate aptamers that display high selectivity for the target thus allowing to easily discriminate between even very close molecules. For instance, RNA aptamers with high selectivity have been generated that bind with nanomolar affinities the protein kinase C, a potential target in cancer medicine, and are capable of discriminating between the P II from the highly related P I isoenzymes (17). DNA aptamers have been obtained that recognize both the native and the denatured state of ERK-2, a member of the family of mitogen-activated protein kinases, which are central transducers of extracellular signals (18). RNA ligands with high affinity for the Ras-binding domain of Raf-1 have been isolated and shown to inhibit either Ras binding to Raf-1 and Ras-induced Raf-1 activation, but they did not affect the interaction of Ras with B-Raf, a Raf-1 related protein (19). Furthermore, highly specific aptamer has been generated against platelet-derived growth factor that suppress platelet-derived growth factor B-chain but not the epidermal- or fibroblast-growth-factor-2-induced proliferation (20).

Among the proteins that have been reported as targets of biomedical interest for development of aptamers as therapeutics, particularly relevant is Tenascin-C (TN-C). It is a large, extracellular matrix glycoprotein that is overexpressed during tissue remodeling such as wound healing, atherosclerosis, psoriasis, and tumor growth (21). To isolate oligonucleotide that can target anticancer drug delivery, selection yielded an aptamer that bound to TN-C with a Kd of 5 nM (22). The TN-C aptamer is currently being developed for tumor-imaging applications (23).

Furthermore, two aptamers have been selected against the prostate-specific membrane antigen (PSMA) overexpressed by the prostate cancer cells (24). The aptamers inhibited the target with a Ki of 2.1 and 11.9 nM. One of the aptamers has been truncated and fluorescently end-labeled to evaluate its ability to bind PSMA-expressing cancer cells. The aptamer bound to LNCaP cells but not

PC-3, showing its specificity for PSMA and its potential in therapeutic development for this target.

Other proteins for aptamer targeting are: human epidermal growth factor receptor-3 (ErbB3/HER3; [25J) and the IFN-y-inducible CXCL10 chemokine (26). In the latter case, Marro et al. (26) identified a series of nuclease-resistant RNA aptamers with high binding affinity for human and/or mouse CXCL10. CXCL10 is a chemokine involved in a variety of inflammatory diseases. Because some of the aptamers are highly selective for CXCL10, they represent a powerful tool to further elucidate the complex cross-talk between the CXCL10/ CXCR3 receptor and other chemokine/receptor system.

As reported in Table 1, several aptamers are actually in clinical trials (27) and the Food and Drug Administration has recently approved one aptamer developed by Eyetech (Macugen™) that inhibits the human vascular endothelial growth factor 165 (VEGF165), for the treatment of age-related macular degeneration (28). In addition to preventing ocular neovascularization, a logical potential therapy for aptamers to VEGF is in cancer. The aptamer isolated and optimized by Ruckman et al. (28) was tested in a mouse model of Wilm's tumor or nephroblastoma (29). Renal histopathology revealed an 84% reduction in tumor weight in the aptamer-treated kidneys compared to the controls. Furthermore, lung metastases were seen in 20% of the aptamer-treated mice compared to 60% of control animals. The aptamer was also tested in a murine model of neuroblastoma, where it resulted in 53% reduction in tumor growth compared to control (30).

Even though a large number of aptamers have been selected for preferential targeting of extracellular proteins or protein epitopes the use of living cells as complex target has been recently described to develop a differential whole-cell SELEX protocol to target cell-surface-bound proteins in their natural physiological environment (31). The selection procedure was performed by using as target the RETC634Y mutant expressed on PC12 cells. A library of 2'-F RNAs was incubated with parental PC12 cells to remove aptamers that bind nonspecifically to the cell surface. To select for aptamers that specifically bound the mutant receptor, the supernatant was incubated with PC12-RETC634Y cells. Unbound sequences were washed off, the whole process reiterated 16 times and the bound winning sequences cloned. The resulting aptamers did not bind to a recombinant EC C634Y RET fragment highlighting the strength of the whole-cell approach. Among the selected aptamers, the best inhibitor (D4) binds specifically to the Ret receptor tyro-sine kinase and blocks its downstream signaling effects on cell differentiation and transformation (31). The results suggest that the differential whole-cell SELEX approach will be useful in the isolation of other lead therapeutic compounds and diagnostic cell-surface markers.

Table 1

Therapeutic Aptamers in Cancer Treatment


Aptamer activity

Therapeutic application in vitro in vivo

Macugen® Inhibition of VEGF165

PDGF-B Inhibition of aptamer PDGF binding to PDGFR ProMune™ Agonist for Toll-like receptor 9 (TLR 9)

Agro 100 Binding to nucleolin

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