3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Inactivation of hepatitis B virus CCCDNA using engineered transcription activator-like affector nucleases(2014-03-31) Bloom, Kristie MichelleHepatitis B virus (HBV) is a major global public health burden, with over 350 million people chronically infected. This results in approximately 600,000 liver cancer-related deaths annually. Chronic HBV infections are normally managed with long-term anti-HBV therapeutics, such as reverse transcription inhibitors, which target post-transcriptional viral processes without affecting the cccDNA. Treatment failure however is largely as a result of the stability of this episomal viral DNA. The cccDNA minichromosome serves as a reservoir of HBV DNA and is capable of re-establishing viral replication following withdrawal of treatment. Designer nucleases, like transcription activator-like effector nucleases (TALENs), have recently been used to create double stranded breaks (DSBs) at target-specific endogenous DNA loci. These nucleases are designed as pairs, which upon dimerisation cleave double-stranded DNA. Subsequent activation of the cellular non-homologous end-joining (NHEJ) pathway often results in targeted mutagenesis at the DSB site. As TALENs may be designed to bind to any DNA sequence, they are commonly used as genetic engineering agents. Inactivation of HBV cccDNA, using these engineered TALENs, presents a unique approach to disabling viral replication permanently. To investigate this, a panel of TALENs targeting the core (C), surface (S) and two different polymerase (P1 and P2) regions of HBV cccDNA were generated using a Golden gate modular assembly approach. TALENs were initially tested in two liver-derived cell lines. Firstly as transient co-transfections in Huh7 cells using a HBV replication competent plasmid, followed by long term investigations in HepG2.2.15 cells which model HBV replication in vitro. Inactivation of HBV was determined by measuring markers of viral replication, whilst TALEN-mediated targeted disruption was verified by T7 endonuclease 1 (T7E1) or CELI endonuclease assays and sequencing. In vitro, the S TALEN inhibited HBsAg secretion by 80% in Huh7 cells and 60% in HepG2.2.15 cells. Furthermore, S TALEN-mediated targeted disruption occurred in 35-47% of cccDNA copies, whilst the C TALEN resulted in 11% targeted disruption of cccDNA in without inhibition of HBsAg expression. The P2 TALEN showed no anti-HBV efficacy, however the P1 TALEN inhibited HBsAg expression by up to 60% without any evidence of site-directed cleavage. As this TALEN binding site spans the HBV Enhancer I sequence, knock-down of HBsAg expression is most likely to occur as a result of transient transcriptional repression. To confirm whether permanent repression of HBV transcription could be achieved, a KRAB-based transcription activator-like repressor (rTALE) targeting the HBV pre-S2 promoter was generated. Using an in vitro reporter gene assay, the pre-S2 rTALE inhibited luciferase expression by up to 90%. However this was only achieved using high molar concentrations of the repressor, suggesting multiple rTALEs may improve HBV transcriptional repression. As the S and C TALENs displayed significant anti-HBV efficiency in vitro, they were tested in a murine hydrodynamic injection model of HBV replication. In vivo, the S TALEN inhibited HBsAg secretion by 95% and induced disruption in 77–87% of HBV DNA targets. In addition the C TALEN inhibited HBcAg expression and induced disruption in 78-93% of HBV DNA targets. Additionally, serological analysis showed a reduction in circulating virions and no apparent liver toxicity, as determined by real-time PCR (qPCR) and aspartate transaminase (AST)/ alanine aminotransferase (ALT) liver function tests respectively. Deep sequencing at the S and C TALEN binding sites showed targeted mutagenesis of HBV DNA in samples extracted from murine hepatocytes transfected with TALENs, however wild-type sequences were exclusively detected in mice that had not been treated with anti-HBV TALENs. Furthermore, frameshift deletions were predominantly detected indicating major disruptions in the HBV surface and core sequences. These results indicate that TALENs designed to disable and silence HBV cccDNA are effective both in vitro and in vivo and as such provide a promising therapeutic approach to treat this serious infection.Item The design and application of a real-time PCR assay to assess rcDNA and cccDNA produced by HBV during infection(2010-08-30) Bloom, Kristie MichelleChronic hepatitis B virus (HBV) infection is endemic to sub-Saharan Africa, and despite the availability of anti-viral agents, there is currently no cure. This double stranded DNA virus is hepatotropic, and active viral replication results in two genomic equivalents, the relaxed circular DNA (rcDNA) and covalently closed circular DNA (cccDNA). The virion encapsulated rcDNA contains a partially synthesised positive DNA stand and a gap region within the negative strand. After infection of hepatocytes, the rcDNA is repaired in the nucleus to form cccDNA. An important objective of HBV therapy is the elimination of cccDNA, as its persistence within hepatocytes has been attributed to chronic HBV infection. Therefore a reliable assay for this replication intermediate is crucial. The objective of this study was to develop a method based on real-time PCR to detect and quantify HBV cccDNA. PCR primers which flank the rcDNA gap were designed to amplify cccDNA whilst primers flanking the pre-S1 region quantify total HBV DNA. Viral DNA was extracted from HepG2.2.15 cells, along with serum and livers from HBV transgenic mice. According to this assay, cccDNA was readily detectable in transgenic mouse livers, but was present at low concentrations in serum samples. The intrahepatic HBV DNA profile of transgenic mice was found to be 40% cccDNA to 60% rcDNA. In HepG2.2.15 cells, only 2% of HBV DNA was cccDNA whilst the majority was in the form of rcDNA. These results were validated using non-radioactive Southern blothybridisation. Additionally, it was established that although RNAi-based effecters inhibit HBV replication, established cccDNA pools were not eliminated. Real-time PCR provides a convenient platform for HBV cccDNA detection as it allows for the rapid simultaneous amplification and quantification of a specific DNA target through either non-specific or specific DNA detection chemistries. In conclusion, this HBV qPCR assay should enable improved monitoring of patients’ responses to antiviral therapy