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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 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.

SUBMITTER: Kennedy EM

PROVIDER: S-EPMC4323668 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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

Kennedy Edward M EM Bassit Leda C LC Mueller Henrik H Kornepati Anand V R AVR Bogerd Hal P HP Nie Ting T Chatterjee Payel P Javanbakht Hassan H Schinazi Raymond F RF Cullen Bryan R BR

Virology 20141229

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 c ...[more]

PMID: 25553515

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Project description:BACKGROUND:Demineralized dentin matrix (DDM) is commonly used as a bone-graft substitute. This study measured and compared human hepatitis B viruses (HBV) DNA in fresh dentin to that of dentin processed into DDM extracted during dental treatment from HBV-infected patients. The hypothesis was that the processing procedure for DDM would inactivate or eliminate HBV in the dentin matrix obtained from infected patients. METHODS:Dentin from eighteen HBV-infected patients was collected and each dentin specimen was divided into two fragments. One fragment was used before processing as fresh dentin (control group) and the other was processed into DDM (experimental group). DNA was extracted and purified from each fresh and processed dentin specimen and the HBV DNA copy number quantitated by real time polymerase chain reaction. The HBV DNA copy number in the fresh dentin specimens were compared relative to serologic test results. The second parameter was to evaluate the effectiveness of the processing procedure (defatting, demineralization, freeze-drying, and sterilization) to inactivate or eliminate HBV by comparing the DNA copy number in the processed DDM with that in the matched fresh dentin specimens. All results were analyzed using Mann-Whitney U test to compare numerical measurements between groups and differences were considered statistically significant at P-values less than 0.05. RESULTS:The presence of HBV DNA was detected in 55.56% (10/18) of the fresh dentin specimens. For the ten HBV DNA-positive fresh dentin specimens, HBV DNA was detected in two (20%) of the matched processed dentin specimens. The copy number of HBV DNA in the two positive processed dentin specimens was 1.79 and 4.03, which were statistically lower than that of the fresh dentin specimens (P?=?0.0167). CONCLUSIONS:The results from this study suggested that fresh dentin may be a carrier of HBV and that the procedure used to generate DDM extensively reduced the levels of HBV DNA. Further studies are needed to evaluate the infectivity of HBV in processed dentin.

Dataset Information

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

Publications

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

Similar Datasets

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