Wolfgang Walther

Gene Therapy of Solid Cancers

Aptamers are an emerging class of molecules in cancer therapy. They can be easily synthesized and are considered cost-effective drug candidates. In this book chapter we describe the selection and characterization of DNA aptamers specifi c to the human epidermal growth factor receptor 2 (ERBB2/HER2), an oncogenic tyrosine kinase. First, a DNA aptamer library is applied and ERBB2-specifi c aptamers are selected using SELEX. Binders are subcloned into a pGEM-T vector, sequenced, and characterized using biochemical and cell biological techniques. By multimerizing the selected ERBB2 aptamers, it might be possible to signifi cantly increase their avidity. For example, we could show that a trimeric ERBB2-specifi c aptamer could effi ciently internalize membranal ERBB2. Furthermore, the receptor assembled in cytoplasmic puncta and was fi nally degraded by the lysosome. In addition, the selected, trimeric aptamer inhibited proliferation in an XTT assay in comparison to a control sequence. Aptamers selected using the protocol we describe might exert anticancer effect. In our example of a trimeric anti-HER2 aptamer, we could report that a human gastric xenograft mouse model demonstrated pharmacological value of the selected aptamer in vivo. This chapter should enable the interested reader to replicate selection of DNA aptamers specifi c to oncogenic cell surface. We would like to particularly emphasize some experimental approaches which were used to further characterize selected aptamer sequences, upon SELEX selection. For instance, we included several blotting techniques, antiproliferative assays of aptamers in vitro, and describe the handling of an in vivo human xenograft mouse model.