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本帖最后由 StephenW 于 2015-4-19 07:02 编辑
RS-2077
Viral hepatitis
Hepatitis B & D - Experimental
TARGETING VIRAL DNA WITH CRISPR/CAS9 ROBUSTLY SUPPRESSES HEPATITIS B VIRUS
Amir Shlomai* 1, 2, 3, Vyas Ramanan4, David B. Cox4, 5, 6, Robert E. Schwartz7, 8, 9, Eleftherios Michailidis1, Ankit Bhatta1, Feng Zhang4, 5, 10, Sangeeta N. Bhatia5, 7, 8, 11, 12, Charles M. Rice1
1Virology and Infectious Disease, Rockefeller University, New-York, United States, 2The Liver Institute, Rabin Medical Center, Beilinson hospital, Petah Tikva, 3Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel, 4Department of Health Sciences and Technology, Massachusetts Institute of Technology, 5Broad Institute, 6Department of Biology, Massachusetts Institute of Technology, 7Department of Health Sciences and Technology, MIT, Cambridge, 8Department of Medicine, Brigham and Women's Hospital, Boston, 9Division of Gastroenterology and Hepatology, Weill Cornell Medical College, New-York, 10Department of Brain and Cognitive Science, 11Department of Electrical Engineering and Computer Science, 12Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
Corresponding author’s email: [email protected]
Background and Aims:
Hepatitis B virus is a 3.2kb DNA virus that infects the liver. Current anti-viral drugs efficiently suppress viral replication but do not clear viral episomal DNA (cccDNA). As a result, lifelong treatment is often needed to control viremia, underscoring the need for drugs or strategies that can effectively eliminate cccDNA and lead to a durable cure. The recent discovery of CRISPR (clustered regularly interspersed short palindromic repeat) as a bacterial adaptive immune system, and subsequent engineering of this system to precisely cleave DNA, provides a potential approach for direct targeting of HBV DNA in infected human hepatocytes.
Results:
We first engineered a set of CRISPR/Cas9 guide RNAs targeting conserved regions in the HBV genome and screened for their utility in-vitro and in-vivo. Hepatoma cells were co-transfected with HBV expressing as well as CRISPR/Cas9 encoding plasmids and animals were hydrodynamically injected with those plasmids to analyze the effect on HBV gene expression and replication. We identified three guide RNAs highly potent for HBV inhibition that were further tested in HepG2215 cells, which stably express HBV from an integrated transgene and also maintain a stable pool of cccDNA. A kinetic analysis revealed a robust reduction in HBV replicative forms as well as in cccDNA levels over time. Analysis of CRISPR-mediated DNA cleavage revealed substantial cleavage presence in integrated HBV DNA but much less in residual cccDNA. This suggests that that cccDNA targeted by CRISPR/Cas9 may be rapidly degraded upon cleavage rather than repaired. Importantly, predicted off-target gene loci were not cleaved, further supporting the mechanism and specificity of this approach. Finally, hepatoma cells over expressing the HBV receptor NTCP and selected for the expression of HBV specific CRISPR/Cas9 guide RNAs were infected with HBV. A large attenuation of HBV infection was observed in CRISPR/Cas9 expressing cells, confirming the utility of this system in the context of natural infection, as well
Conclusions:
The CRISPR/Cas9 system suppresses HBV replication and possibly eliminates cccDNA. Our study provides a proof of concept for targeting DNA viruses with CRISPR/Cas9 and highlights the possible utility of this approach as a curative anti-HBV therapy.
Disclosure of Interest: None Declared
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发表于 2015-4-19 06:56
