Generating obligate heterodimeric transcription activator-like effector nucleases to inactive hepatitis B viral replication
Date
2018
Authors
Smith, Tiffany Shenay
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Abstract
The hepatitis B virus (HBV) remains a global health concern as chronic infection can lead to cirrhosis and hepatocellular carcinoma. Current therapies are only partially effective and do not target the HBV covalently closed circular DNA (cccDNA). cccDNA serves as a replication intermediate for the transcription of viral RNA, resulting in the persistence of infection. Several designer nucleases have therefore been developed to target cccDNA. Their mutagenic effect is mediated by causing double-strand breaks at target sites, consequently disrupting translation of viral proteins. Previous studies observed successful inhibition of HBV, with the use of homodimeric transcription activator-like effector nucleases (TALENs). TALENs are designed in pairs (left and right monomers) to cleave complementary strands of targeted DNA. They consist of a DNA-binding domain which is attached to the wildtype nuclease domain of FokI. TALENs have exhibited considerable predictable interaction with their target sites and have shown diminished toxicity in vitro and in vivo. Despite this success, TALENs containing wildtype FokI nuclease domains may be prone to off-target effects as two of the same domains (two left or two right) in close proximity on opposite strands of DNA can result in cleavage at unintended sites. To establish a more clinically applicable therapy, obligate heterodimeric TALENs targeted against the core (C) and surface (S) open reading frames (ORFs) of cccDNA were generated and referred to as C or S Variant TALENs. Variant TALENs contain mutant FokI nuclease domains. This allows for cleavage only when two different domains (one left and one right) are in close proximity on opposite strands of DNA thus reducing off-target effects. Overall the variant TALENs demonstrated significant knockdown of the HBV replication marker, hepatitis B surface antigen (HBsAg). Additionally variant TALEN-mediated targeted disruption occurred in ≥5.7% of total HBV DNA in vitro and ≥13% of total HBV DNA in vivo. Finally variant TALEN-treated mice also demonstrated significant knockdown of pregenomic RNA and surface mRNA levels as well as markedly reduced circulating viral particle equivalents. Together these results show that obligate heterodimeric TALENs are capable of HBV knockdown and serve as an advanced gene editing tool against chronic HBV infection.
Description
A dissertation submitted to the Faculty of Health Sciences, University of the
Witwatersrand, in fulfilment of the requirements for the degree
of
Master of Science in Medicine
Johannesburg, 2018.