title: Improved antiviral efficacy using TALEN-mediated homology directed recombination to introduce artificial primary miRNAs into DNA of hepatitis B virus.
abstract: Chronic infection with hepatitis B virus (HBV) remains an important global health problem. Currently licensed therapies have modest curative efficacy, which is as a result of their transient effects and limited action on the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Gene editing with artificial HBV-specific endonucleases and use of artificial activators of the RNA interference pathway have shown anti-HBV therapeutic promise. Although results from these gene therapies are encouraging, maximizing durable antiviral effects is important. To address this goal, a strategy that entails combining gene editing with homology-directed DNA recombination (HDR), to introduce HBV-silencing artificial primary microRNAs (pri-miRs) into HBV DNA targets, is reported here. Previously described transcription activator-like effector nucleases (TALENs) that target the core and surface sequences of HBV were used to introduce double stranded breaks in the viral DNA. Simultaneous administration of donor sequences encoding artificial promoterless anti-HBV pri-miRs, with flanking arms that were homologous to sequences adjoining the TALENs' targets, augmented antiviral efficacy. Analysis showed targeted integration and the length of the flanking homologous arms of donor DNA had a minimal effect on antiviral efficiency. These results support the notion that gene editing and silencing may be combined to effect improved inhibition of HBV gene expression.
authors: SA-MRC/Wits Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa.
authors: Timothy
authors: T
authors: Dreyer
authors: SA-MRC/Wits Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa.
authors: Samantha
authors: S
authors: Nicholson
authors: SA-MRC/Wits Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa.
authors: Abdullah
authors: A
authors: Ely
authors: SA-MRC/Wits Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa.
authors: Patrick
authors: P
authors: Arbuthnot
authors: SA-MRC/Wits Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa. Electronic address: Kristie.Bloom@wits.ac.za.
authors: Kristie
authors: K
authors: Bloom
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics*; D056945:Hep G2 Cells; D006514:Hepatitis B Surface Antigens / Q000378:metabolism; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000187:drug effects; D000069896:Transcription Activator-Like Effector Nucleases / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D035683:MicroRNAs; D000069896:Transcription Activator-Like Effector Nucleases
keywords: Artificial primary miRNA; HBV; TALEN; cccDNA