title: Knockdown of a G protein-coupled receptor through efficient peptide-mediated siRNA delivery.
abstract: In recent years, therapeutic applications of siRNAs have come into the focus of pharmaceutical research owing to their potential to specifically regulate gene expression. However, oligonucleotides have to overcome a series of extracellular and intracellular barriers which is why delivery systems helping to overcome these barriers are desperately needed. A promising approach to transport nucleic acids beyond cellular membranes is the use of cell-penetrating peptides (CPPs), which are able to autonomously cross the plasma membrane. Recently, we synthesized branched derivatives of truncated human calcitonin (hCT) and identified them as efficient vehicles for non-covalent gene delivery. Here we describe two novel branched hCT-derivatives that are optimized for efficient intracellular delivery of siRNA by conjugation with either a fatty acid or an endosomolytic peptide sequence. As target we chose the human NPY Y₁ receptor (NPY1R), which belongs to the family of G protein-coupled receptors and thus constitutes a model for complex therapeutic targets related to various disorders. For instance, knockdown of Y₁ receptor expression offers a potential therapy for osteoporosis. We present a read-out system that allows for the quantitation of the induced knockdown of receptor expression on the protein as well as on the mRNA level. As a result of this study, we could show that the herein presented cell-penetrating peptides effectively transport siRNA into HEK-293 cells without inducing cytotoxicity and that the knockdown rates are comparable to those obtained by lipofection.
authors.affiliation: Translational Centre for Regenerative Medicine, University of Leipzig, Philipp-Rosenthal-Str. 55, D-04103 Leipzig, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Hoyer
authors.forename: Ines
authors.initials: I
authors.lastname: Neundorf
mesh_terms: D002116:Calcitonin / Q000235:genetics*; D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000008:administration & dosage*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D034741:RNA, Small Interfering / Q000008:administration & dosage*; D017476:Receptors, Neuropeptide Y / Q000235:genetics*; D014162:Transfection / Q000379:methods*
chemical_list: D057846:Cell-Penetrating Peptides; D034741:RNA, Small Interfering; D017476:Receptors, Neuropeptide Y; C097212:neuropeptide Y-Y1 receptor; D002116:Calcitonin