Investigating the enzymatic processing and gene knockdown of micelle-conjugated siRNA against glioblastoma

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Event Abstract Back to Event Investigating the enzymatic processing and gene knockdown of micelle-conjugated siRNA against glioblastoma Amy Arnold1*, Christopher Mclaughlin2, Saul Martinez-Montero3, Elise Malek-Adamian3, Christopher Kent4*, Masad Damha3, Kevin Petrecca4 and Molly Shoichet1, 2 1 University of Toronto, Chemistry, Canada 2 University of Toronto, Chemical Engineering, Canada 3 McGill University, Chemistry, Canada 4 McGill University, Neurology and Neurosurgery, Canada Introduction: Glioblastoma (GBM), a type of brain cancer, is one of the deadliest cancers and represents a significant clinical challenge. It is hypothesized that GBM cancer stem cells (GSCs) are the major cell type responsible for glioblastoma invasion and recurrence.[1] Small interfering ribonucleic acids (siRNAs) can be engineered to target specific proteins responsible for cancer cell proliferation and invasion.[2] However, these nucleic acids are susceptible to cleavage in vivo. Polymeric nanomicelles are promising vehicles for the delivery of genetic therapeutics to cancerous tissue, stabilizing the nucleic acids with respect to cleavage and enabling targeted delivery strategies.[3] As an additional stabilization strategy, Damha and coworkers have synthesized nucleic acids that substitute 2′-ribose for 2′-fluoro-ribose (FRNA), 2′-fluoro-β-D-arabinose (FANA) or β-D-arabinose (ANA) in the oligonucleotide chain.[4] These modifications increase the intracellular half-life of the resulting siRNA.[5] siRNAs that are synthesized as Dicer substrates (an important enzyme for siRNA cell trafficking) show enhanced efficacy.[6] The Dicer processing of siRNA-polymer systems has been examined for poly(ethylene glycol)[7] and lipid[8] conjugates, but these studies are scarce. Importantly, the Dicer processing of fluoro-modified siRNAs has not been directly investigated. In this work, the Dicer processing of fluoro-modified, micelle-conjugated siRNA will be presented. The gene knockdown in GSCs is also investigated using these dicer-substrate siRNAs. A gene associated with the GSC invasion, resistance, and stemness was chosen as the genetic target in a series of primary GSC lines.[9],[10] Materials and Methods: Oligonucleotides are synthesized using standard phosphoramidite chemistry. Micelles are synthesized as previously described[3] and the siRNA-micelle conjugation is carried out by a Huisgen click type reaction between the DBCO-functionalized oligonucleotide and the azide-functionalized micelle. Dicer enzyme kit was used as purchased from Genlantis. The electrophoretic mobility of oligonucleotides and digested products were analyzed using native polyacrylamide electrophoresis. Individual gel lanes were quantified using density measurements obtained with Image J software. Gene knockdown studies were performed using Lipofectamine 2000 (purchased from Invitrogen) in a 6-well format. Results and Discussion: siRNA duplexes were designed with varying substitution patterns (unmodified, partly modified, and fully modified). Dicer processing was conducted on each siRNA duplex, both unconjugated and conjugated to the micelle, and digested products analyzed by native PAGE. Significant processing was observed in all cases, though the unmodified strands were observed to undergo the most enzyme cleavage. Significant gene knockdown was observed when transfecting the siRNA into glioblastoma cells, both unconjugated and conjugated to the micelle. Conclusions: The results of this study indicate that Dicer is still able to effectively process modified siRNA, even with the duplexes conjugated to a micelle. In addition, the siRNA sequences developed here were able to regulate gene expression in glioblastoma stem cells. Looking ahead, these results are a promising indicator of siRNA potency and their incorporation into a targeted nanomicelle delivery system for GBM therapy is currently under investigation. National Science and Engineering Research Council of Canada; Canadian Institutes of Health ResearchReferences:[1] Singh, S. K.; Hawkins, C.; Clarke, I. D.; Squire, J. A.; Bayani, J.; Hide, T.; Henkelman, R. M.; Cusimano, M. D.; Dirks, P. B. Identification of Human Brain Tumour Initiating Cells. Nature 2004, 432, 396–401.[2] Anzahaee, M. Y.; Deleavey, G. F.; Le, P. U.; Fakhoury, J.; Petrecca, K.; Damha, M. J. Arabinonucleic Acids: 2'-Stereoisomeric Modulators of siRNA Activity. Nucleic Acid Ther 2014, 24, 336–343[3] Chan, D. P. Y.; Deleavey, G. F.; Owen, S. C.; Damha, M. J.; Shoichet, M. S. Click Conjugated Polymeric Immuno-Nanoparticles for Targeted siRNA and Antisense Oligonucleotide Delivery. Biomaterials 2013, 34, 8408–8415.[4] Watts, J. K.; Damha, M. J. 2′F-Arabinonucleic Acids (2′F-ANA) — History, Properties, and New Frontiers. Can. J. Chem. 2008, 86, 641–656.[5] Ferrari, N.; Bergeron, D.; Tedeschi, A.-L.; Mangos, M. M.; Paquet, L.; Renzi, P. M.; Damha, M. J. Characterization of Antisense Oligonucleotides Comprising 2“-Deoxy-2-”Fluoro-Beta-D-Arabinonucleic Acid (FANA): Specificity, Potency, and Duration of Activity. Ann. N. Y. Acad. Sci. 2006, 1082, 91–102.[6] Kim, D.-H.; Behlke, M. A.; Rose, S. D.; Chang, M.-S.; Choi, S.; Rossi, J. J. Synthetic dsRNA Dicer Substrates Enhance RNAi Potency and Efficacy. Nat. Biotechnol. 2005, 23, 222–226.[7] Kow, S. C.; McCarroll, J.; Valade, D.; Boyer, C.; Dwarte, T.; Davis, T. P.; Kavallaris, M.; Bulmus, V. Dicer-Labile PEG Conjugates for siRNA Delivery. Biomacromolecules 2011, 12, 4301–4310.[8] Kubo, T.; Takei, Y.; Mihara, K.; Yanagihara, K.; Seyama, T. Amino-Modified and Lipid-Conjugated Dicer-Substrate siRNA Enhances RNAi Efficacy. Bioconjugate Chem. 2012, 23, 164–173.[9] Dudley, A.; Sater, M.; Le, P. U.; Trinh, G.; Sadr, M. S.; Bergeron, J.; Deleavey, G. F.; Bedell, B.; Damha, M. J.; Petrecca, K. DRR Regulates AKT Activation to Drive Brain Cancer Invasion. Oncogene 2014, 33, 4952–4960.[10] Suvà, M. L.; Rheinbay, E.; Gillespie, S. M.; Patel, A. P.; Wakimoto, H.; Rabkin, S. D.; Riggi, N.; Chi, A. S.; Cahill, D. P.; Nahed, B. V.; et al. Reconstructing and Reprogramming the Tumor-Propagating Potential of Glioblastoma Stem-Like Cells. Cell 2014, 157, 580–594. Keywords: stem cell, biomaterial, siRNA delivery, vehicle Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Enabling RNAi with biomaterials Citation: Arnold A, Mclaughlin C, Martinez-Montero S, Malek-Adamian E, Kent C, Damha M, Petrecca K and Shoichet M (2016). Investigating the enzymatic processing and gene knockdown of micelle-conjugated siRNA against glioblastoma. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02142 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. * Correspondence: Dr. Amy Arnold, University of Toronto, Chemistry, Toronto, ON, Canada, Email1 Dr. Christopher Kent, McGill University, Neurology and Neurosurgery, Montreal, ON, Canada, Email2 Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Amy Arnold Christopher Mclaughlin Saul Martinez-Montero Elise Malek-Adamian Christopher Kent Masad Damha Kevin Petrecca Molly Shoichet Google Amy Arnold Christopher Mclaughlin Saul Martinez-Montero Elise Malek-Adamian Christopher Kent Masad Damha Kevin Petrecca Molly Shoichet Google Scholar Amy Arnold Christopher Mclaughlin Saul Martinez-Montero Elise Malek-Adamian Christopher Kent Masad Damha Kevin Petrecca Molly Shoichet PubMed Amy Arnold Christopher Mclaughlin Saul Martinez-Montero Elise Malek-Adamian Christopher Kent Masad Damha Kevin Petrecca Molly Shoichet Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.