Helper-dependent adenoviral vectors expressing anti-HBV pri-miR sequences from the liver-specific PEPCK promoter
Date
2017
Authors
Smit, Duodane
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Abstract
Hepatitis B is a global health problem that kills about 600 000 people annually. It is an infectious disease caused by the Hepatitis B Virus (HBV), which infects the liver and leads to liver inflammation and secondary complications such as cirrhosis and Hepatocellular Carcinoma (HCC). The available therapies are only partially effective and are associated with adverse side effects and viral drug resistance. RNA interference (RNAi) pathway is a gene silencing pathway found in diverse living systems including mammals. Harnessing of this pathway to inhibit HBV replication has shown a lot of promise, with highly effective anti-HBV RNAi activator sequences designed. However, the lack of safe and efficient delivery and expression systems for these sequences is one of the obstacles that need to be overcome before RNAi effectors can reach clinical application. For easy assessment of transduction efficiency, Helper Dependent Adenoviral vectors (HDAd) expressing β-galactosidase (encoded by lac Z gene) are commonly used to deliver anti-viral RNAi activators. However, this reporter protein has been blamed for induction of innate immune response and concomitant clearance of the HDAds by the host. For the first time, this study explored the use of lac Z-deficient HDAds to deliver anti-HBV RNAi activators expressed under the control of a liver-specific phosphoenolpyruvate carboxykinase (PEPCK) promoter. HDAd expressing Firefly luciferase resulted in a significant luminescence detected in cell culture lysates and a sustained bioluminescence in mice. HDAds expressing anti-HBV sequences transduced the liver efficiently and did not induce a pronounced inflammatory response or liver toxicity in mice. However, this did not translate into maximal anti-HBV sequence expression and HBV replication inhibition in vitro and in vivo. This suggests that the PEPCK promoter is inadequate for RNAi activator expression. This study highlights the importance of careful RNAi activator regulatory elements selection and presents the therapeutic potential and utility of HDAd vectors for hepatotropic delivery of antiviral sequences with markedly attenuated immune response stimulation and toxicity. For improved anti-HBV RNAi activator expression and HBV knockdown, a different liver specific promoter mouse transthyretin receptor (MTTR) promoter is currently being investigated in our lab.
Description
A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree
of
Master of Science in Medicine
Johannesburg, 2017