Targeted inactivation of hepatitis B virus genome using enhanced transcription activator-like effector nucleases containing a sharkey fokI nuclease domain

• Main Author: Singh, Prashika
• Format: Others
• Language: en
• Published: 2018
• Online Access: https://hdl.handle.net/10539/25431

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. === Despite the availability of an effective vaccine, hepatitis B virus (HBV) infections are a major public health problem with approximately two thirds of the world’s population having been exposed to this virus. This DNA virus infects hepatocytes causing inflammation and is the causative agent of hepatitis B. Upon infection the viral genome is transported to the nucleus and repaired to covalently closed circular DNA (cccDNA). The cccDNA intermediate is responsible for the persistence of HBV in chronically infected individuals and acts as a viral reservoir. Current treatments are not able to target the stable cccDNA and highlight the need for the development of new therapies. This project focused on the development of anti-HBV transcription activator-like effector nucleases (TALENs) with improved efficiency and safety. TALENs are designer nucleases that induce double-stranded breaks (DSBs) within DNA. This stimulates host repair pathways that results in insertions or deletions (indels) that cause frame-shift mutations and disrupt targeted genes. TALENs are highly promising as they are able to target cccDNA and could lead to a functional cure for HBV. Existing anti-HBV TALENs have been designed to target conserved sites in the S and C open reading frames of the HBV genome. The third generation obligate heterodimeric Sharkey variants are enhanced FokI nuclease domains that were generated by altering certain amino acid residues in the wild-type FokI nuclease domain. Sharkey TALENs were produced by substituting the wild-type FokI domain of existing anti-HBV TALENs with Sharkey variants. In vitro analysis of the anti-HBV Sharkey TALENs demonstrated expression of the nucleases in liver-derived cells. However, the wild-type anti-HBV TALENs exhibited superior inhibitory effects as compared to the Sharkey TALENs. Sequence-specific cleavage was achieved by both surface (S) and core (C) Sharkey TALENs however the latter achieved greater targeted cleavage. Mutagenesis analysis of HBV DNA from extracted HepG2.2.15 cells revealed the Sharkey C TALEN achieved 12.1% targeted cleavage greater compared to the Sharkey S TALEN. Efficacy was assessed in vivo in the mouse hydrodynamic injection model of HBV replication. In vivo viral markers of HBV replication were assessed and the Sharkey anti-HBV S and C TALENs were able to reduce viral surface antigen secretion by 57% and 78% respectively, whereas both Sharkey TALENs achieved a 96% reduction of circulating viral particle equivalents. The Sharkey TALENs were able to reduce viral mRNA production by 35% and 45% and pregenomic RNA levels by 73% and 80%. Minimal toxicity in vitro and in vivo was observed using the Sharkey TALENs indicating that reduction of markers of HBV replication were as a consequence of the action of the Sharkey anti-HBV TALENs. Results for the Sharkey C TALEN were comparable to the wild-type TALENs in vitro and in vivo. Despite the inhibition of HBV replication achieved with third generation Sharkey TALENs, a more comprehensive analysis of anti-HBV Sharkey TALEN activity in different in vitro and in vivo models of HBV replication is necessary. In addition, in depth analysis of potential off-target effects is required to comprehensively examine the safety of these variants. Nonetheless, in this study the Sharkey TALENs are able to inhibit HBV replication cccDNA and transcription, thereby further validating the use of TALENs as a potential therapy for chronic HBV infection. === LG2018