PepFect6 Mediated SiRNA Delivery into Organotypic Cultures
Gene silencing by small interfering RNA (SiRNA) is an attractive therapeutic approach for pathological disorders that targets a specific gene. However, its applications are limited, as naked RNA is rapidly degraded by RNases and is inadequately internalized by the target cells in the body. Several viral and nonviral vectors have been described to improve the delivery of SiRNAs both in cultured cells as well as in vivo. Increasing evidence suggests that cell-penetrating peptides (CPPs) are an efficient, non-cytotoxic tool for intracellular delivery of SiRNA. Recently, a new peptide, PepFect6 (PF6), based system has been described for efficient SiRNA delivery in various cell types. PF6 is an amphipathic stearyl-TP10 peptide carrying a pH titratable trifluoromethylquinoline moiety that facilitate endosomal release. PF6 forms stable non-covalent complexes with SiRNA. Upon internalization, the complexes rapidly escape the endosomal compartment, resulting in robust RNA interference (RNAi) responses. This chapter describes a protocol to use the PF6-nanoparticle technology for SiRNA delivery into organotypic cultures of the inner ear i.e., cochlea. We also highlight different critical points in the peptide/SiRNA complex preparation, transfection and in analyzing the efficacy of PF6-SiRNA associated RNAi response.
Key wordsCell-penetrating peptides (CPPs) PepFect6 (PF6) SiRNA Inner ear Cochlea Organotypic cultures Fluorescence recovery after photobleaching (FRAP)
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