Tumor-targeting of therapeutic oligonucleotides with DARPins

The modulation of single gene functions by RNA interference, above all using small interfering RNA (siRNA), has reached increasing importance in basic life science research during the last decade. The use for targeted knockdown of single disease-relevant genes promises great benefit for medical applications, particularly for cancer therapy. However, all efforts so far as well as experiences with a structurally very closely related compound class, the antisense oligonucleotides, have identified the difficulties that have to be overcome for successful therapeutic application. In particular, the problem of delivering the nucleic acid to its intracellular target has not yet been solved satisfactorily, because the inherent biological properties of these molecules hamper cell membrane permeation, and negatively influence bioavailability and cellular uptake. In addition, high specificity, stability, and prevention of immune activation are issues that must be addressed in order to develop molecular drugs tailored for maximum specificity and efficiency. In this transnational research project, a novel tumor-targeted delivery system for therapeutic nucleic acids will be developed and optimized by careful evaluation in preclinical models in vitro and in vivo. This nanomedicine makes use of the superior functional properties of DARPins (Designed Ankyrin Repeat Proteins) as tumor-targeting ligands. Upon binding to EpCAM as our selected tumor-associated antigen, the complexed or chemically attached antisense and siRNA molecules are delivered specifically into tumor cells by receptor-mediated endocytosis. In previous work we have demonstrated the proof-of-principle of using DARPins for tumor cell-targeted siRNA delivery. In the proposed project, the delivery system will be further optimized in terms of affinity, loading capacity, and intracellular release of the nucleic acid payload upon internalization. DARPins will be evolved to higher affinity using ribosome display selection as well as a multimerization strategy (gain of avidity). Attachment of the nucleic acids will be optimized by evaluation of different binding and complexing strategies. Finally, we will employ novel chemical oligonucleotide modifications designed to improve plasma stability and cell membrane permeation, and to reduce off-target effects. The nanomedicine will be tested in tumor models in vitro and in vivo, including system biological methods, to gather information about the properties required to produce effective cancer therapeutics, and identify drug candidates for further preclinical and clinical investigations. This is a joint project of a Swiss group, leading in engineering protein ligands for tumor-targeting and an Austrian group with comprehensive experience in development and optimization of therapeutic nucleic acids.

The joint research project was concerned with the development of DARPin-based carrier systems for application of therapeutic oligonucleotides. Both antisense and siRNA technologies promise great progress in therapy of various diseases, including cancer, viral infections and autoimmune diseases. Triggered by sequence-specific target recognition, any desired gene function can be inhibited, and thus, therapeutically active oligonucleotides are suited to regulate previously unavailable therapeutic targets. However, several unsolved challenges, comprising mainly bioavailability (transport to the intracellular site of action), stability and cell type specificity, need to be overcome in order to fulfill the promise. Designed Ankyrin Repeat Proteins are artifical binding proteins with affinities similar to antibodies, but their smaller size, simpler structure, high stability and the lack of posttranslational modifications facilitate recombinant production and derivatization for complexation or coupling of effector entities. Therefore, they are perfectly suited as carrier molecules for specific transport into tumor cells. We developed a carrier system that is suited for receptor-mediated targeted delivery into cancer cells. We generated fusion proteins consisting of a target-recognizing DARPin component and a basic peptide for charge-complexing oligonucleotides. All components of the fusion proteins were optimized separately for target affinity and oligonucleotide capacity, respectively. Our research showed that these fusion proteins aggregate to nanoparticular structures with complexed oligonucleotides. The biochemical properties such as particle size, stability in solution, and cell type affinities were examined and optimized. A tetrameric DARPin format consisting of two distinct binding motifs (to the same receptor) and a short, arginine-rich peptide, proved to be the most appropriate approach. In cell culture models we could show that specific uptake nearly exclusively into antigen-positive tumor cells took place with oligonucleotides causing target down regulation of the corresponding gene.