Nucleic acid aptamers for target validation and therapeutic applications

Abstract

In the simplest view, aptamers can be thought of as nucleic acid analogs to antibodies. They are able to bind specifically to proteins, and, in many cases, that binding leads to a modulation of protein activity. New aptamers are rapidly generated through the SELEX (Systematic Evolution of Ligands by Exponential enrichment) process and have a very high target affinity and specificity (picomoles to nanomoles). Furthermore, aptamers composed of modified nucleotides have a long in vivo half-life (hours to days), are nontoxic and nonimmunogenic, and are easily produced using standard nucleic acid synthesis methods. These properties make aptamers ideal for target validation and as a new class of therapeutics. As a target validation tool, aptamers provide important information that complements that provided by other methods. For example, siRNA is widely used to demonstrate that protein knock-out in a cellular assay can lead to a biological effect. Aptamers extend that information by showing that the dose-dependent modulation of protein activity can be used to derive a therapeutic benefit. That is, aptamers can be used to demonstrate that the protein is a good target for drug development. As a new class of therapeutics, aptamers bridge the gap between small molecules and biologics. Like biologics, biologically active aptamers are rapidly discovered, have no class-specific toxicity, and are adept at disrupting protein-protein interaction. Like small molecules, aptamers can be rationally engineered and optimized, are nonimmunogenic, and are produced by scalable chemical procedures at moderate cost. As such, aptamers are emerging as an important source of new therapeutic molecules.

FIGURE 1

Diagram outlining the SELEX process.

FIGURE 2

The levels of gene expression and corresponding target validation techniques.

FIGURE 3

Treatment with platelet-derived growth factor receptor (PDGFr) antagonists lowers interstitial fluid pressure in KAT-4 tumors. Tumor interstitial fluid pressure was measured 1–2 h after last administration of PDGF receptor inhibitor in KAT-4 tumors grown subcutaneously in severe combined immune deficiency (SCID) mice. PDGF receptor inhibitors were administered for a total of 4 days. A: Mice were treated with phosphate buffered saline (PBS) (n = 8) or STI571 (n = 9). B: Mice were treated with control aptamer (n = 8) or PDGF aptamer (n = 8). (Reproduced with permission from Pietras et al. Cancer Res 2002;62: 5476–5484.)

FIGURE 4

Treatment with PDGF receptor antagonists enhances the effect of Taxol on KAT-4 tumors in vivo. Growth curves of KAT-4 tumors grown subcutaneously in SCID mice. A: Mice received no treatment (solid square, n = 8), STI571 (solid circle, n = 6), Taxol (solid triangle, n = 4), or STI571 and Taxol (cross bar, n = 8). B: Mice received polyethylene glycol (PEG; solid square, n = 8), PEG-conjugated PDGF aptamer (solid circle, n = 8), PEG and Taxol (solid triangle, n = 8), or PDGF aptamer and Taxol (cross bar, n = 8). *P < 0.05, PDGF-receptor antagonist and Taxol versus Taxol alone, Student’s t-test, **P < 0.01 PDGF-receptor antagonist and Taxol versus Taxol alone, Student’s t-test. (Reproduced with permission from Pietras et al. (Cancer Res 2002;62: 5476–5484.)

FIGURE 5

Treatment with PDGF aptamer antagonists increases uptake of [³H]Taxol in KAT-4 tumors. After treatment with PDGF-receptor antagonists or control, mice with KAT-4 tumors were injected subcutaneously with [³H] Taxol. Radioactivity was measured in homogenates of tumors and in blood samples 8 or 24 h after subcutaneous injection of radiolabeled compound. Mice were treated with control aptamer (8 h, n = 6; 24 h, n = 7) or PDGF aptamer (8 h, n = 6; 24h, n = 7); *P < 0.05, Student’s t test; **P < 0.01, Student’s t-test; bars, ± SE. (Reproduced with permission from Pietras et al. Cancer Res 2002;62:5476–5484.)