Small interfering RNAs (siRNAs) are specific and effective molecules for gene silencing, but fail to enter cells unassisted due to their negative charges and their high molecular weight/size. The conjugation of siRNA to ligands targeting cell surface receptors is a promising approach to silence genes associated with various pathologies. However, besides the external cell barrier, to reach the cytosol where the siRNA associates with the RISC (RNA-induced silencing complex) machinery, the siRNA has to escape from intracellular compartments (ICC). However, despite encouraging perspectives, RNA therapeutic applications are hindered by a low rate of ICC escape. To enhance the cytosolic delivery of siRNAs, one strategy is based on multi-ligand conjugates. Its efficacy has been proven with recently marketed drugs composed of three N-acetylgalactosamine ligands conjugated to siRNA. This bioconjugate has led the way for the use of siRNA conjugates in therapy. However, these conjugates are not transposable to pathologies other than liver diseases. Moreover, mechanisms of escape across ICC remain unknown, the underlying mechanisms relating affinity/avidity/specificity to siRNA cytosolic delivery remain to be elucidated, and active targeting strategies for organs/cells other than liver/hepatocytes, remain to be explored. Our project aims at deciphering and optimizing the delivery of siRNAs mediated by aptamers. Aptamers are nucleic acid molecules which bind to their targets with high affinity and selectivity. When the target is a cell-surface biomarker, aptamers have huge potential as specific cell-targeting ligands. If the receptor is internalized, aptamers can drive conjugated siRNA inside cells. Moreover, their chemical synthesis allows for a variety of modulable constructs. Our project aims to provide strategies based on multivalent/multispecific nucleic acid aptamers as innovative active targeting tools to enhance selective siRNA delivery to targeted cells. We will develop multifunctional molecules, in which the aptamer(s) is/are the ‘cell-targeting part(s)’ and the siRNA is the ‘therapeutic part’. These aptamers conjugated to siRNA are named AsiC for aptamer-siRNA conjugates. This project, based on one siRNA and three aptamers targeting three different cell surface receptors, aims to 1) synthesize conjugates composed of 1-3 aptamers conjugated to siRNA, 2) characterize their interaction kinetics, and 3) follow their intracellular trafficking, to in fine provide insights to improve the delivery of siRNA by active targeting.