title: Silencing of hepatitis C virus replication by a non-viral vector based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry site (IRES).
abstract: Gene silencing mediated by RNAi has gained increasing interest as an alternative for the treatment of infectious diseases such as refractory hepatitis C virus (HCV) infection. In this work we have designed and evaluated a non-viral vector based on solid lipid nanoparticles (SLN) bearing hyaluronic acid, protamine and a short hairpin RNA (shRNA74) targeted to the Internal Ribosome Entry Site (IRES) of the HCV. The vector was able to inhibit the expression of the HCV IRES in Huh-7 cells, with the inhibition level dependent on the shRNA74 to SLN ratio and on the shRNA74 dose added to the culture cells. The nanocarrier was also able to inhibit the replication in human hepatoma cells supporting a subgenomic HCV replicon (Huh-7 NS3-3'). The vector was quickly and efficiently internalized by the cells, and endocytosis was the most productive uptake mechanism for silencing. Clathrin-mediated endocytosis and to a lesser extent caveolae/lipid raft-mediated endocytosis were identified as endocytic mechanisms involved in the cell uptake. Internalization via the CD44 receptor was also involved, although this entry route seems to be less productive for silencing than endocytosis. The vector did not induce either hemolysis or agglutination of red cells in vitro, which was indicative of good biocompatibility. In summary, we have shown for the first time the ability of a non-viral SLN-based vector to silence a HCV replicon.
authors.affiliation: Pharmacokinetic, Nanotechnology and Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigación Lascaray ikergunea, University of the Basque Country UPV/EHU, Paseo de la Universidad, 7. 01006, Vitoria-Gasteiz, Spain. Electronic address: josune.torrecilla@ehu.eus.
authors.forename: Josune
authors.initials: J
authors.lastname: Torrecilla
authors.affiliation: Pharmacokinetic, Nanotechnology and Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigación Lascaray ikergunea, University of the Basque Country UPV/EHU, Paseo de la Universidad, 7. 01006, Vitoria-Gasteiz, Spain. Electronic address: ana.delpozo@ehu.eus.
authors.forename: Ana
authors.initials: A
authors.lastname: Del Pozo-Rodríguez
authors.affiliation: Pharmacokinetic, Nanotechnology and Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigación Lascaray ikergunea, University of the Basque Country UPV/EHU, Paseo de la Universidad, 7. 01006, Vitoria-Gasteiz, Spain. Electronic address: marian.solinis@ehu.eus.
authors.forename: María Ángeles
authors.initials: MÁ
authors.lastname: Solinís
authors.affiliation: Pharmacokinetic, Nanotechnology and Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigación Lascaray ikergunea, University of the Basque Country UPV/EHU, Paseo de la Universidad, 7. 01006, Vitoria-Gasteiz, Spain. Electronic address: paolastephanie.apaolaza@ehu.eus.
authors.forename: Paola S
authors.initials: PS
authors.lastname: Apaolaza
authors.affiliation: Instituto de Parasitología y Biomedicina "López-Neyra" (IPBLN-CSIC), PTS Granada, Av del conocimiento s/n, Armilla, 18016, Granada, Spain. Electronic address: bbh@ipb.csic.es.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Berzal-Herranz
authors.affiliation: Instituto de Parasitología y Biomedicina "López-Neyra" (IPBLN-CSIC), PTS Granada, Av del conocimiento s/n, Armilla, 18016, Granada, Spain. Electronic address: cristina_romero@ipb.csic.es.
authors.forename: Cristina
authors.initials: C
authors.lastname: Romero-López
authors.affiliation: Instituto de Parasitología y Biomedicina "López-Neyra" (IPBLN-CSIC), PTS Granada, Av del conocimiento s/n, Armilla, 18016, Granada, Spain. Electronic address: aberzalh@ipb.csic.es.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Berzal-Herranz
authors.affiliation: Pharmacokinetic, Nanotechnology and Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigación Lascaray ikergunea, University of the Basque Country UPV/EHU, Paseo de la Universidad, 7. 01006, Vitoria-Gasteiz, Spain. Electronic address: alicia.rodriguez@ehu.eus.
authors.forename: Alicia
authors.initials: A
authors.lastname: Rodríguez-Gascón
mesh_terms: D000998:Antiviral Agents; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000821:virology; D002470:Cell Survival; D017930:Genes, Reporter; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000235:genetics / Q000628:therapy*; D006801:Humans; D000067556:Internal Ribosome Entry Sites / Q000235:genetics*; D008055:Lipids / Q000737:chemistry*; D008113:Liver Neoplasms / Q000235:genetics / Q000821:virology; D053758:Nanoparticles / Q000008:administration & dosage* / Q000737:chemistry; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D012093:Replicon; D014407:Tumor Cells, Cultured; D014779:Virus Replication / Q000235:genetics*
chemical_list: D000998:Antiviral Agents; D000067556:Internal Ribosome Entry Sites; D008055:Lipids; D034741:RNA, Small Interfering
keywords: HCV-IRES; Hepatitis C; Hyaluronic acid; RNAi; Solid lipid nanoparticles; shRNA