title: Harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated Cas9 system to disrupt the hepatitis B virus.
abstract: The current therapies to treat hepatitis B virus (HBV) infection are limited. Recently, clustered regularly interspaced short palindromic repeat (CRISPR) systems, originally identified in bacteria and archaea, have been found to consist of an RNA-based adaptive immune system that degrades complimentary sequences of invading plasmids and viruses. Here, we studied the effects of the CRISPR/CRISPR-associated Cas9 system that was targeted to the surface antigen (HBsAg)-encoding region of HBV, both in a cell culture system and in vivo. The HBsAg levels in the media of the cells and in the sera of mice were analyzed by a quantitative enzyme-linked immunosorbent assay. The HBV DNA levels were assessed by quantitative PCR and HBsAg expression in mouse livers was assessed by an immunohistochemical assay. The amount of HBsAg secreted in the cell culture and mouse serum was reduced by CRISPR/Cas9 treatment. Immunohistochemistry analyses showed almost no HBsAg-positive cells in the liver tissue of CRISPR/Cas9-S1+X3-treated mice. The CRISPR/Cas9 system efficiently produced mutations in HBV DNA. Thus, CRISPR/Cas9 inhibits HBV replication and expression in vitro and in vivo and may constitute a new therapeutic strategy for HBV infection.
authors: 1] Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing, China [2] Xijing Hospital, Fourth Military Medical University, Xi'an, China [3] Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
authors: S
authors: S
authors: Zhen
authors: 1] Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing, China [2] Laboratory of Pharmacology and Toxicology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
authors: L
authors: L
authors: Hua
authors: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing, China.
authors: Y-H
authors: YH
authors: Liu
authors: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing, China.
authors: L-C
authors: LC
authors: Gao
authors: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing, China.
authors: J
authors: J
authors: Fu
authors: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing, China.
authors: D-Y
authors: DY
authors: Wan
authors: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing, China.
authors: L-H
authors: LH
authors: Dong
authors: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing, China.
authors: H-F
authors: HF
authors: Song
authors: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing, China.
authors: X
authors: X
authors: Gao
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D005260:Female; D015316:Genetic Therapy; D056945:Hep G2 Cells; D006509:Hepatitis B / Q000628:therapy*; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation
chemical_list: D006514:Hepatitis B Surface Antigens
references: N Engl J Med. 1991 Sep 5;325(10):729-31
references: 1651454
references: Hepatobiliary Pancreat Dis Int. 2011 Jun;10(3):295-302
references: 21669574
references: Nat Med. 2010 Mar;16(3):254-7
references: 20208503
references: Hepatology. 2003 Apr;37(4):748-55
references: 12668966
references: Nature. 1980 Jul 31;286(5772):533-5
references: 6250074
references: J Virol. 1983 Aug;47(2):337-43
references: 6620456
references: Nature. 2005 Jun 2;435(7042):646-51
references: 15806097
references: Proc Natl Acad Sci U S A. 2011 Feb 8;108(6):2623-8
references: 21262818
references: Cell Mol Life Sci. 1999 Aug 15;55(10):1304-26
references: 10487208
references: Cell. 2013 Sep 12;154(6):1380-9
references: 23992846
references: Gastroenterology. 2002 Dec;123(6):1831-8
references: 12454840
references: N Engl J Med. 1997 Dec 11;337(24):1733-45
references: 9392700
references: Antiviral Res. 2007 Jan;73(1):24-30
references: 16844238
references: Nat Biotechnol. 2014 Mar;32(3):279-84
references: 24463574
references: World J Gastroenterol. 2007 Jan 7;13(1):125-40
references: 17206760
references: World J Gastroenterol. 2004 Mar 15;10(6):841-6
references: 15040029
references: Nat Biotechnol. 2013 Mar;31(3):230-2
references: 23360966
references: Mol Ther. 2013 Feb;21(2):266-8
references: 23369965
references: J Genet Genomics. 2012 May 20;39(5):209-15
references: 22624882
references: Dev Biol Stand. 1981;50:293-300
references: 6281110
references: Virology. 1990 Mar;175(1):255-61
references: 2155510
references: Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13825-30
references: 12374864
references: PLoS Comput Biol. 2013;9(7):e1003131
references: 23861664
references: Hum Gene Ther. 1999 Jul 1;10(10):1735-7
references: 10428218
references: Vaccine. 2004 Jan 2;22(3-4):439-46
references: 14670326
references: Cell. 2014 Feb 27;156(5):935-49
references: 24529477
references: Proc Natl Acad Sci U S A. 2014 Aug 5;111(31):11461-6
references: 25049410
references: PLoS One. 2013;8(3):e59801
references: 23536890
references: Nat Biotechnol. 2014 Jun;32(6):551-3
references: 24681508
references: Nucleic Acids Res. 2013 Nov;41(20):e187
references: 23997119
references: Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):E2579-86
references: 22949671
references: N Engl J Med. 1982 Jun 10;306(23):1384-7
references: 6281640
references: N Engl J Med. 2003 Feb 27;348(9):808-16
references: 12606735
references: Lancet. 1993 Nov 27;342(8883):1335-40
references: 7901639
references: J Virol. 2012 Sep;86(17):8920-36
references: 22718830
references: Cell. 2013 May 9;153(4):910-8
references: 23643243
references: Nat Rev Mol Cell Biol. 2013 Jan;14(1):49-55
references: 23169466
references: J Hepatol. 2009 Sep;51(3):581-92
references: 19616338
references: J Gastroenterol Hepatol. 2000 Apr;15(4):357-68
references: 10824878
references: Methods Mol Biol. 2010;649:247-56
references: 20680839
references: Cell. 1986 Nov 7;47(3):451-60
references: 3768961
references: Nat Biotechnol. 2013 Mar;31(3):227-9
references: 23360964
references: J Virol. 1994 Mar;68(3):2026-30
references: 8107266
references: Sci Rep. 2013;3:2510
references: 23974631
references: Sci Rep. 2013 Nov 27;3:3355
references: 24284873
references: Mol Ther. 2013 Oct;21(10):1889-97
references: 23883864
references: J Virol. 1996 Jul;70(7):4567-75
references: 8676483
references: Virology. 1982 Aug;121(1):195-9
references: 7112938
references: Nat Biotechnol. 2011 Aug 05;29(8):699-700
references: 21822242
references: J Med Virol. 2005 Jun;76(2):203-7
references: 15834875
references: Nature. 2010 Nov 4;468(7320):67-71
references: 21048762
references: Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1156-60
references: 8577732
references: Science. 2013 Feb 15;339(6121):819-23
references: 23287718
references: Chem Soc Rev. 2011 Dec;40(12):5657-68
references: 21566825
references: Nat Biotechnol. 2013 Jan;31(1):23-4
references: 23302927
references: Annu Rev Med. 2001;52:29-49
references: 11160766
references: Science. 2013 Feb 15;339(6121):823-6
references: 23287722
references: Annu Rev Biochem. 2010;79:213-31
references: 20192761
references: Mol Syst Biol. 2013;9:641
references: 23340847
references: J Cell Physiol. 2008 Aug;216(2):289-94
references: 18302164
references: Semin Immunopathol. 2013 Jan;35(1):39-52
references: 22829332
references: Science. 2012 Aug 17;337(6096):816-21
references: 22745249
references: J Clin Virol. 2005 Dec;34 Suppl 1:S79-82
references: 16461229
references: Hepatology. 2004 Sep;40(3):727-37
references: 15349913