Simultaneous targeting of HCV replication and viral binding with a single lentiviral vector containing multiple RNA interference expression cassettes.
Chronic hepatitis C virus (HCV) infection has a major medical impact and current treatments are often unsuccessful. RNA interference represents a promising new approach to tackling this problem. The current study details the design and testing of self-inactivating lentiviral vectors (LV) delivering RNA interference to prevent HCV replication and infection. Vectors were constructed with single, double, and triple cassettes expressing short hairpin RNAs (shRNAs) simultaneously targeting two regions of the HCV 1b genome and the host cell receptor, CD81. The shRNAs directed against HCV IRES or NS5b regions were shown to be effective in inhibiting HCV replication in vitro (82 and 98%, respectively). No evidence of shRNA-related interferon production was observed. Vectors containing CD81 shRNA reduced cell surface expression up to 83% and reduced cell binding of HCV surface protein E2 up to 82% while not affecting levels of unrelated surface protein (Ber-EP4) or HCV replication. Double or triple shRNA vectors were independently effective in simultaneously reducing HCV replication, CD81 expression, and E2 binding. This study demonstrates lentiviral delivery of multiple shRNA, inhibiting HCV in a specific, IFN-independent, manner. The targeting of multiple viral and host cell elements simultaneously by RNAi could increase the potency of antiviral gene therapies.
Henry
Scot D
SD
Department of Surgery, Erasmus MC-University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.
van der Wegen
Pascal
P
Metselaar
Herold J
HJ
Tilanus
Hugo W
HW
Scholte
Bob J
BJ
van der Laan
Luc J W
LJ
D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D015536:Down-Regulation; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics*; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D007372:Interferons / Q000096:biosynthesis; D016086:Lentivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D034622:RNA Interference; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D015703:Antigens, CD; C554798:CD81 protein, human; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus; D007372:Interferons