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Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression

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Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression Research Article Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression Manuela Aleku,1 Petra Schulz,2 Oliver Keil,1 Ansgar Santel,1 Ute Schaeper,1 Britta Dieckhoff,1 Oliver Janke,1 Jens Endruschat,1 Birgit Durieux,1 Nadine Ro¨der,1 Kathrin Lo¨ffler,1 Christian Lange,1 Melanie Fechtner,1 Kristin Mo¨pert,1 Gerald Fisch,1 Sibylle Dames,1 Wolfgang Arnold,1 Karin Jochims,3 Klaus Giese,1 Bertram Wiedenmann,2 Arne Scholz,2 and Jo¨rg Kaufmann1 1Silence Therapeutics AG; 2Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Charite´-Universita¨tsmedizin, Berlin, Germany; and 3IASON Consulting, Niederzier, Germany Abstract of chemically synthesized siRNA for therapeutic purposes (for a We have previously described a small interfering RNA (siRNA) technology review, see refs. 2, 4, 6–8). With respect to cancer, a delivery system (AtuPLEX) for RNA interference (RNAi) in the variety of nonviral nanoparticles (50–200 nm) have been recently vasculature of mice. Here we report preclinical data for reported to be suitable for RNAi-mediated tumor growth inhibition Atu027, a siRNA-lipoplex directed against protein kinase N3 in experimental mouse models (summarized in ref. 6). In most (PKN3), currently under development for the treatment of cases, various siRNA carriers such as cyclodextrin (transferrin advanced solid cancer. In vitro studies revealed that Atu027- receptor targeted; ref. 9), PEI (RGD-targeted PEG-PEI; ref. 10), mediated inhibition of PKN3 function in primary endothelial DOTAP (self-assembled LPD-nanoparticle; nanosized immunolipo- cells impaired tube formation on extracellular matrix and some; refs. 11, 12), AtuFECT (AtuPLEX; refs. 5, 13), or other cationic cell migration, but is not essential for proliferation. Systemic lipids (14–17) have been developed for efficient complexation and administration of Atu027 by repeated bolus injections or delivery of siRNAs. The addition of targeting ligands (transferrin, infusions in mice, rats, and nonhuman primates results in anisamide, RGD-peptide, poly-arginine, anti-TfR single-chain anti- specific, RNAi-mediated silencing of PKN3 expression. We body fragment/TfRscFv, DNA) and additional lipid moieties show the efficacy of Atu027 in orthotopic mouse models for (PEG-lipids, cholesterol, endosomolytic helper lipids, or peptides) prostate and pancreatic cancers with significant inhibition or the overall morphology of the generated nanoparticles of tumor growth and lymph node metastasis formation. The (characterized by charge and particle size) supposedly confer tumor vasculature of Atu027-treated animals showed a specific targeting specificity to either cancer cells and/or tumor vascula- reduction in lymph vessel density but no significant changes in ture. Likewise, siRNAs targeting the expression of oncogenes microvascular density. [Cancer Res 2008;68(23):9788–98] (EphA2, EWS-Fli-1, Raf-kinase, EGFR/Her2) or angiogenesis-pro- moting genes (VEGFR2 and CD31) were chosen for RNAi-mediated Introduction therapeutic intervention in oncology. It remains debatable by which mechanism siRNA-containing RNA interference (RNAi) can be used as novel therapeutic nanoparticles (>60 nm) can escape the circulation and cross the modality through specific silencing of therapeutically relevant endothelial barrier to efficiently penetrate the tumor tissue. genes in vivo (1–3). In particular, chemically synthesized, small However, in contrast to other organ parenchyma, the vascular interfering RNAs (siRNA) are currently used as a new class of bed (endothelial cells) is directly accessible for systemically therapeutic molecules, allowing the controlled down-regulation of administered particles and therefore predestinated to endocytosis pathologically relevant gene expression as for oncogenes in cancer of siRNA-loaded particles, offering the reasonable approach for (4). However, in analogy to classic antisense molecules the in vivo RNAi-based therapeutic modulation of the vasculature. Hence, an application of these molecules is not straightforward. The overall antiangiogenic approach by means of ‘‘formulated siRNA-inhibi- negative charge of siRNA molecules (up to 40 negative charges) and tor’’ targeting the tumor vascular compartment might bear the relatively high molecular weight (12,000–14,000 Da) prevent valuable strategic and therapeutic advantages. these from functional uptake of these novel therapeutic molecules We recently described a novel liposomal siRNA formulation in vivo. Besides the inefficient uptake and the degradation in based on cationic lipids (siRNA-lipoplex/AtuPLEX), containing endosomal compartments on the cellular level, unformulated neutral fusogenic and PEG-modified lipid components, for siRNAs are rapidly cleared by renal excretion from the blood improved pharmacokinetic properties, cellular uptake, and effi- stream when administered i.v. (5). cient siRNA release from the endosomes after endocytosis. Using To overcome these limitations, delivery technologies for this formulation, we have shown a selective inhibition of gene synthetic siRNAs are currently being developed, enabling the use expression in endothelial cells in vivo as shown by target-specific knockdown of CD31 [platelet endothelial cell adhesion molecule-1 (PECAM-1)] or TIE-2 in various mouse tissues (5, 18). Moreover, we Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). showed therapeutic efficacy in xenograft mouse tumor models Requests for reprints: Jo¨rg Kaufmann, Silence Therapeutics AG, Otto-Warburg after systemic administration of CD31 targeting siRNA-lipoplexes Haus, Robert-Ro¨ssle-Strasse 10, Berlin 13125, Germany. Phone: 49-30-9489-2833; (13). The observed efficacy was not related to toxic side effects Fax: 49-30-9489-2801; E-mail: [email protected]. I2008 American Association for Cancer Research. including unspecific stimulation of the innate immune system. doi:10.1158/0008-5472.CAN-08-2428 Repeated dosing of the AtuPLEX does not elevate systemic levels Cancer Res 2008; 68: (23). December 1, 2008 9788 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. Atu027 for RNAi-Based Cancer Therapy of cytokines such as IFN-a or interleukin (IL)-12 (5, 13). These Supplementary Table S1. All AtuPLEXes (siRNA-lipoplexes) were prepared results laid the foundation for the preclinical development of this as described previously (5). The physicochemical properties of Atu027 novel liposomal siRNA formulation for RNAi therapeutics in particles are summarized in Supplementary Fig. S1. In vitro oncology. transfection, immunoblotting, and functional assays. The siRNA-lipoplex containing 0.28 mg/mL siRNA (19 Amol/L) was further Here we discuss preclinical data on Atu027, a lipoplexed siRNA diluted to a final concentration of 1 to 20 nmol/L siRNA in 10% serum– molecule specifically targeting the expression of protein kinase N3 containing cell culture medium. Human HeLa, PC-3, and murine EOMA cell (PKN3). PKN3 has previously been identified as a downstream lines were obtained and cultivated according to the American Type Culture effector of the phosphoinositide-3-kinase (PI3K) signaling pathway Collection recommendation. Human umbilical vascular endothelial cells (19). Loss of PKN3 function in vascular and lymphatic endothelial (HUVEC) and human microvascular endothelial lung lymphatic cells cells by RNAi suggested PKN3 as an appropriate molecular target (HMVEC-LLy) were obtained from Lonza. for an antiangiogenesis therapeutic strategy. We show the thera- The following antibodies were used for immunoblotting: rabbit anti- peutic potential of Atu027 for tumor treatment in orthotopic mouse PTEN (Cell Signaling), rabbit anti-PKN3 (19), mouse anti–Hsp60 (BD tumor models along with PKN3 gene silencing in vivo. Furthermore, Laboratories), rabbit anti–Akt-1 (Cell Signaling), and mouse anti-tubulin we noticed changes in the tumor lymphatic vasculature on (Oncogene). For the Matrigel assay, HUVEC and HMVEC-LLy cells were Atu027 treatment, suggesting an anti-lymphangiogenesis mode of trypsinized 72 h posttransfection, counted, and seeded on Matrigel action for the inhibition of tumor growth and metastasis. basement membrane matrix (Becton Dickinson) with a seeding density of 100,000 per 24-well plate. At the same time point, cells were lysed for Western blotting to measure PKN3 protein expression. Photographs were Materials and Methods taken after 6 h to evaluate tube formation. In the transmigration (Boyden siRNA molecules and preparation of siRNA-lipoplexes. siRNA chamber) assay, HUVEC cells were trypsinized 72 h posttransfection, molecules (AtuRNAi, blunt-ended 23-mer, alternating 2¶-O-methyl modified) counted, and seeded onto Fluoroblok inserts in serum-free medium. Bottom were previously described (20) and were synthesized by BioSpring. To wells were filled with serum- and growth factor–containing medium as screen for PKN3 mRNA inhibition, eight different mouse and human chemoattractant. At the same time point, cells were lysed for Western specific siRNA molecules were designed by bioinformatics means with the blotting to show PKN3 knockdown. After 6 h, migrated cells were visualized human/mouse-specific PKN3(3) lead siRNA sequence, sense strand 1552- by staining with SYTOX Green (Molecular Probes), and photographs were agacuugaggacuuccuggacaa-1574 (NM_013355.3); for the rat studies, a rat taken to
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