Targeting HBV cccDNA with Viral Vector Carrying HBV-Specific CRISPR/Cas9
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Alrashed, Bayan
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Abstract
Chronic HBV infection is a worldwide public health concern. It is characterized by the persistence of the hepatitis B surface antigen (HBsAg) in serum for more than six months. The persistence of HBsAg is mediated by viral covalently closed circular DNA (cccDNA) in the nuclei of infected cells. Although several genome-editing techniques have demonstrated the possibility to inhibit HBV viral replication, attempts to completely clear nuclear cccDNA remain unsuccessful. Recent studies have suggested the possibility to use the Clustered Regularly Interspaced Short Palindromic Repeats system (CRISPR/Cas9) to suppress HBV infection; however, effective delivery of this tool for in vivo targeting is a major issue. To examine the possibility to utilize the CRISPR/Cas9 system for HBV clearance, we plan to generate a mouse model of chronic HBV infection by using a recombinant Adeno-associated virus that expresses HBV (rAAV-HBV). It has been previously suggested that a rAAV-HBV virus can establish persistent HBV infection in mice. We would like to use this model to test whether viral vectors that carry HBV-specific CRISPR/Cas9 can target HBV cccDNA in vivo. To this end, the recombinant AAV-HBV virus was produced by an AAV helper free expression system in HEK293T cells. As a control, I also produced a rAAV-GFP virus from co-transfection of HEK293T cells. My results show that the recombinant AAV-GFP virus can effectively infect human liver HepG2 cells and express high levels of green fluorescent protein. I also examined the rAAV-HBV virus for expressing HBV genes in HepG2 cells by western blot and quantitative real-time PCR (qPCR). While western blots showed defined bands of HBsAg in HepG2 cells, qPCR of infected HepG2 culture supernatants and cell lysates yielded a low amount of HBV genomes. We therefore demonstrated the possibility to use rAAV-HBV vector to delivery HBV genome into liver cells. However, the efficiency is relatively low, likely resulting from the large size of the HBV genome that may affect the AAV packaging efficiency. Multiple approaches to improve the efficiency of the rAAV-HBV vector are currently being tested.
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This document has been embargoed for 5 years and will not be available until August 2021 at the earliest.
Keywords
Hepatitus B virus, CCC DNA, Adeno-associated virus, CRISPR/cas9, Viral vector, Lenti-viral vector