Extracellular Targets

The majority of aptamers with potential therapeutic utility selected to date target extracellular proteins. Extracellular therapeutic targets, such as growth and coagulation factors of the vasculature, have the advantage of ready access to aptamer intervention without need for enabling aptamer access to cells or tissue spaces. Broad therapeutic areas are represented among aptamers directed against extracellular targets, including angiogenesis/oncology (Green et al., 1995, 1996; Nobile et al., 1998; Ruckman et al., 1998; Lupold et al., 2002; Chen et al., 2003; White et al., 2003), inflammation (Wiegand et al., 1996; Rhodes et al., 2000, 2001), anticoagulation and thrombosis (Bock et al., 1992; Gal et al., 1998; Rusconi et al., 2004; Rusconi et al., 2000, 2002; Tasset et al., 1997), and autoimmune disease (Tsai and Keene, 1993; Doudna et al., 1995; Lee and Sullenger, 1997; Kim et al., 2003). The aptamer drug Macugen (pegaptanib) (Ruckman et al., 1998; Eyetech Study Group, 2002, 2003), which is approved for treatment of age-related macular degeneration (AMD), targets vascular endothelial growth factor (VEGF), a key mediator of an-giogenesis, by inhibiting VEGF binding to the receptor tyrosine kinases KDR and Flt-1 (Ruckman et al., 1998).

VEGF blockade for treatment of colorectal cancer has been clinically validated with a monoclonal antibody (Ferrara, 2004), and one can envision a similar application for anti-VEGF aptamers such as pegaptanib. Basic fibroblast growth factor (bFGF), like VEGF, is a mediator of angiogenesis that acts through FGF receptor-1 (FGFR-1), a receptor tyrosine kinase expressed on the endothelial cell surface. A high-affinity anti-bFGF aptamer inhibited receptor binding, bFGF-dependent en-dothelial cell migration and proliferation (Jellinek et al., 1995). Angiopoietin-2 (Ang-2) is an angiogenic factor that potentiates the pro-angiogenic activity of VEGF and bFGF on endothelial cells by acting as a Tie2 receptor tyrosine kinase antagonist (Lobov et al., 2002). White and co-workers showed that an anti-Ang-2 aptamer modulates Tie2 activity in vitro and, more significantly, inhibited bFGF-induced corneal angiogenesis in a rat model (White et al., 2003).

The first aptamer of potential therapeutic value was selected against thrombin (Bock et al., 1992) soon after the advent of SELEX (Tuerk and Gold, 1990). Subsequently, inhibitors for several other components of the coagulation cascade have been generated (Tasset et al., 1997; Gal et al., 1998; Rusconi et al., 2000, 2002; Jeter et al., 2004). Complex formation between factor VIIa (FVIIa) and tissue factor (TF) in response to vascular injury initiates the extrinsic arm of the coagulation cascade. Rusconi et al. (2002) showed that an aptamer highly specific for FVIIa inhibited coagulation in vitro, most likely by blocking complex formation between FVIIa and TF. Coagulation factor IXa (FIXa) is a component of the intrinsic clotting cascade that activates factor X. Rusconi and co-workers developed an aptamer (9.3t) to FIXa that suppresses factor X activation and is a potent anticoagulant in vitro (Rusconi et al., 2002). Moreover, the activity of 9.3t can be reversed by a complementary oligonucleotide antidote both in vitro and in vivo (Rusconi et al., 2004).

Notable extracellular proteins that play a role in inflammation include cytokines, chemokines, and antibodies. Wiegand et al. (1996) selected aptamers to IgE, a mediator of local inflammation which can cause allergies, atopic dermatitis, and asthma when overexpressed in atopic individuals (Sutton and Gould, 1993). Allergen-specific IgE binds to its high-affinity receptor, FceR1, on the surface of mast cells and basophils, which in turn activates the cells and results in degranulation and release of proinflammatory mediators. IgE-directed aptamers block IgE binding to FceR1, and inhibit degranulation in vitro. In another application, Rhodes et al. (2000) sought an inhibitor of oncostatin M (OSM), a potent proin-flammatory cytokine that may be a key mediator of rheumatoid arthritis (Plater-Zyberk et al., 2001). The authors showed that an aptamer to OSM specifically inhibited OSM receptor binding and receptor activation in vitro. The same group developed an aptamer against MCP-1, a ß/CC chemokine whose levels are elevated in serum from patients with asthma and rheumatoid arthritis (Rhodes et al., 2001). Mouse MCP-1 was used as the bait for SELEX to enable testing of the resulting aptamers in murine disease models. The resulting aptamers were shown to be potent inhibitors of mouse MCP-1-dependent chemotaxis in vitro.

Several studies have probed the potential of aptamers for the treatment of antibody-mediated autoimmune disease (Tsai and Keene, 1993; Doudna et al., 1995;

Lee and Sullenger, 1996; Lee and Sullenger, 1997; Hwang et al., 2003; Kim et al., 2003). In each case, aptamers that recognize and block the activity of specific autoantibodies were generated. Extreme insulin resistance type B is mediated by auto-antibodies to a main antigenic epitope on the surface of the insulin receptor (Zhang and Roth, 1991). Antibody binding to the receptor induces internalization, or downmodulation, of the receptor (Roth et al., 1983). A mouse monoclonal antibody, mAb20, which recognizes a primary antigenic epitope on the human insulin receptor, was successfully used as a SELEX target (Doudna et al., 1995; Lee and Sullenger, 1996). The resulting aptamers blocked mAb20 binding to the insulin receptor, and prevented antibody-mediated, but not insulin-mediated receptor in-ternalization (Lee and Sullenger, 1996). Interestingly, one aptamer cross-reacted with patient autoimmune serum and was able to block receptor downregulation induced by patient serum in vitro.

Myasthenia gravis (MG) is a neuromuscular disorder that results from an antibody-mediated autoimmune response to the nicotinic acetylcholine receptor (AChR) (Drachman, 1994). Hwang and co-workers selected an aptamer to a rat anti-AChR antibody, mAb198, which is cross- reactive with the human receptor and is also able to induce experimental autoimmune myasthenia gravis (EAMG) in rats (Hwang et al., 2003). The aptamer, tMG, blocked mAb198-depen-dent AChR downregulation on the surface of TE671 cells, and was also cross-reactive with serum from a MG patient. A polyethylene glycol (PEG)-conjugated (i.e. PEGylated) version of the aptamer, PEG-tMG, improved the clinical score in rats in the mAb198-induced EAMG model. Rats treated with PEG-tMG had higher muscle AChR content than either untreated rats, or rats treated with unPEGylated or scrambled versions of PEG-tMG. Although the results of these and other studies are intriguing, the utility of this treatment modality may actually be limited by the heterogeneity of autoimmune antibodies present in patient sera and the exquisite specificity of aptamers.

Osteoarthritis

Osteoarthritis

Thank you for deciding to learn more about the disorder, Osteoarthritis. Inside these pages, you will learn what it is, who is most at risk for developing it, what causes it, and some treatment plans to help those that do have it feel better. While there is no definitive “cure” for Osteoarthritis, there are ways in which individuals can improve their quality of life and change the discomfort level to one that can be tolerated on a daily basis.

Get My Free Ebook


Post a comment