Delivery of Immunostimulatory RNA Oligonucleotides by Gelatin Nanoparticles Triggers an Efficient Antitumoral Response Carole Bourquin,* Cornelia Wurzenberger,* Simon Heidegger,* Sebastian Fuchs,w David Anz,z Sarah Weigel,* Nadja Sandholzer,* Gerhard Winter,w Conrad Coester,w and Stefan Endres* Summary: RNA oligonucleotides have emerged as a new class of biologicals that can silence gene expression but also stimulate immune responses through specific pattern-recognition receptors. The development of effective delivery systems remains a major challenge for the therapeutic application of the RNA oligonucleo- tides. In this study, we have established a novel biodegradable carrier system that is highly effective for the delivery of immunostimulatory RNA oligonucleotides. Formulation of RNA oligonucleotides with cationized gelatin nanoparticles potentiates immune activation through the Toll-like receptor 7 (TLR7) in both myeloid and plasmacytoid dendritic cells. Further, nanoparticle-delivered RNA oligonucleotides trigger production of the antitumoral cytokines IL-12 and IFN-a. Binding to gelatin nanoparticles protects RNA oligonucleotides from degradation by nucleases, facilitates their uptake by dendritic cells, and targets these nucleic acids to the endosomal compartment in which they are recognized by TLR7. In these effects, the nanoparticles are superior to the conventional transfection reagents lipofectamine, polyethylenimine, and DOTAP. In vivo, the delivery of TLR7-activating RNA oligonucleotides by gelatin nanoparticles triggers antigen-specific CD8 + T-cell and antibody responses. Indeed, immunization with RNA-loaded nano- particles leads to an efficient antitumoral immune response in two different mouse tumor models. Thus, gelatin-based nanoparticles represent a novel delivery system for immunostimulatory RNA oligonucleotides that is both effective and nontoxic. Key Words: Toll-like receptor 7, RNA oligonucleotides, tumor immunotherapy, nanoparticles, delivery (J Immunother 2010;33:935–944) U pon viral infection, a strong immune response is elicited through recognition of the viral components by specific receptors of the innate immune system. Molecular patterns within viral nucleic acids are recognized by the pattern- recognition receptors TLR7 (single-stranded RNA se- quences), TLR9 (CpG DNA sequences), RIG-I (retinoid acid inducible gene I, 5 0 -triphosphate RNA), and MDA-5 (double-stranded RNA). 1–5 Synthetic DNA and RNA oligonucleotides containing these molecular patterns are powerful tools to stimulate an immune response in a highly selective manner. It is for instance well established that binding of synthetic CpG DNA oligonucleotides to TLR9 both promotes innate immunity and triggers the generation of a protective Th1-type immune response. 6 In experimental models and in cancer patients, CpG oligonucleotides enhance cytotoxic T-cell responses to tumor antigens upon vaccina- tion. 7–9 Furthermore, we have recently shown that RNA oligonucleotides can stimulate innate immunity through both the Toll-like receptor 7 and RIG-I receptors 3,10 and, thereby induce efficient antitumor responses. 11,12 TLR7-activating RNA oligonucleotides act on several components of both the murine and the human immune systems: they potentiate T-cell and B-cell responses to antigen, 13 activate neutrophils 14 and antitumoral NK cells, 11,15 and block the suppressive function of regulatory T cells. 16 We have shown that these effects are orchestrated by TLR7-expressing dendritic cells that control immune activation through the production of a panel of cyto- kines. 11,13,16 The therapeutic potential of TLR7 agonists is supported by the clinical efficacy of the imidazoquinolines, a class of antitumor agents that acts in part through the activation of TLR7. 17 The lead compound, imiquimod, is approved for the treatment of skin tumors by topical use but is effective against solid tumors only when applied locally. 18 RNA oligonucleotides thus form a new class of TLR7 agonists with promising therapeutic potential. Furthermore, RNA oligonucleotides can be designed to include other antitumoral properties in the same molecule: introduction of an inhibitory siRNA sequence permits silencing of tumor-promoting genes that synergizes with the immunostimulatory activity of the RNA oligonucleotides to block tumor growth. 12 A key challenge for the therapeutic application of the RNA oligonucleotides is the need for efficient in vivo delivery to protect RNA oligonucleotides from degrada- tion, to promote cellular uptake, and to target the RNA to the desired intracellular compartment. 19 In this study, we have investigated the efficacy of cationized gelatin-based nanoparticles for the delivery of immunostimulatory RNA oligonucleotides. Gelatin presents the advantage of being biodegradable and nontoxic and has been used in patients as a plasma expander for decades. 20,21 Gelatin nanoparti- cles are stable during storage, show high stability after administration and can be easily scaled-up for manufactur- ing processes. Highly homogeneous gelatin nanoparticles of Copyright r 2010 by Lippincott Williams & Wilkins Received for publication February 3, 2010; accepted August 1, 2010. From the *Center for Integrated Protein Science Munich and Division of Clinical Pharmacology; Departments of zInternal Medicine, Innenstadt; and wPharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilian University of Munich, Munich, Germany. The authors declared no conflict of interests. Carole Bourquin and Cornelia Wurzenberger have contributed equally to this work. Supported by grants from the German Research Foundation (DFG En 169/7-2 and Graduiertenkolleg 1202 to C.B. and S.E., the excellence cluster CIPSM 114 to S.E.), from LMUexcellent (research professorship to S.E.), from the Else-Kro¨ ner Fresenius Foundation, and from BayImmuNet to C.B. and S.E. This work is part of the doctoral thesis of SF and SW at the Ludwig-Maximilian University of Munich. Reprints: Carole Bourquin, Division of Clinical Pharmacology, Ziems- senstr. 1, 80336 Mu¨ nchen, Germany (e-mail: carole.bourquin@ med.lmu.de). BASIC STUDY J Immunother  Volume 33, Number 9, November–December 2010 www.immunotherapy-journal.com | 935