title: Successful disabling of the 5' UTR of HCV using adeno-associated viral vectors to deliver modular multimeric primary microRNA mimics.
abstract: Chronic hepatitis C virus (HCV) infection is a major health concern and is strongly associated with cirrhosis, hepatocellular carcinoma and liver-related mortality. The HCV genome is the template for both protein translation and viral replication and, being RNA, is amenable to direct genetic silencing by RNA interference (RNAi). HCV is a highly mutable virus and is capable of escaping RNAi-mediated silencing. This has highlighted the importance of developing RNAi-based therapy that simultaneously targets multiple regions of the HCV genome. To develop a multi-targeting RNAi activator, a novel approach for the generation of anti-HCV gene therapy was investigated. Five artificial primary miRNA (pri-miR) were each designed to mimic the naturally occurring monomeric pri-miR-31. Potent knockdown of an HCV reporter was seen with four of the five constructs and were processed according to the intended design. The design of the individual pri-miR mimics enabled the modular assembly into multimeric mimics of any possible conformation. Consequently the four potent pri-miR mimics were used to generate polycistronic cassettes, which showed impressive silencing of an HCV target. To further their application as a gene therapy, recombinant adeno-associated viral (rAAV) vectors that express the polycistronic pri-miR mimics were generated. All AAV-delivered anti-HCV pri-miR mimics significantly knocked down the expression of an HCV target and showed inhibition of HCV replicon replication. Here we describe a protocol for the generation of therapeutic rAAVs that express modular polycistronic pri-miR cassettes allowing for rapid alteration and generation of tailored therapeutic constructs against HCV.
authors.affiliation: Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Science, University of the Witwatersrand, Johannesburg, South Africa.
authors.forename: Tarryn
authors.initials: T
authors.lastname: Bourhill
authors.affiliation: Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Science, University of the Witwatersrand, Johannesburg, South Africa.
authors.forename: Patrick
authors.initials: P
authors.lastname: Arbuthnot
authors.affiliation: Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Science, University of the Witwatersrand, Johannesburg, South Africa. Electronic address: Abdullah.Ely@wits.ac.za.
authors.forename: Abdullah
authors.initials: A
authors.lastname: Ely
mesh_terms: D020121:5' Untranslated Regions; D000256:Adenoviridae / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D018716:Molecular Mimicry; D034622:RNA Interference
chemical_list: D020121:5' Untranslated Regions; D035683:MicroRNAs
keywords: AAV; Gene therapy; HCV; Pri-miRNA mimics; RNA interference