Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease

Abstract Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in...

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Bibliographic Details
Published in:Virology (New York, N.Y.) N.Y.), 2015-02, Vol.476, p.196-205
Main Authors: Kennedy, Edward M, Bassit, Leda C, Mueller, Henrik, Kornepati, Anand V.R, Bogerd, Hal P, Nie, Ting, Chatterjee, Payel, Javanbakht, Hassan, Schinazi, Raymond F, Cullen, Bryan R
Format: Article
Language:English
Subjects:
Antiviral
Bacteria
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
cccDNA
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Associated Proteins - metabolism
CRISPR-Cas Systems
CRISPR/Cas
Deoxyribonuclease I - metabolism
DNA editing
DNA, Viral - genetics
DNA, Viral - metabolism
Down-Regulation
Gene Targeting - methods
HBV
Hepatitis B - therapy
Hepatitis B - virology
Hepatitis B virus
Hepatitis B virus - genetics
Hepatitis B virus - physiology
Humans
Infectious Disease
Lentiviral vector
Streptococcus pyogenes - enzymology
Streptococcus pyogenes - genetics
Virus Replication
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Summary:Abstract Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals.
ISSN:0042-6822
1096-0341
DOI:10.1016/j.virol.2014.12.001