title: A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice.
abstract: Many genetic liver diseases in newborns cause repeated, often lethal, metabolic crises. Gene therapy using nonintegrating viruses such as adeno-associated virus (AAV) is not optimal in this setting because the nonintegrating genome is lost as developing hepatocytes proliferate. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR-Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC). This resulted in reversion of the mutation in 10% (6.7-20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. Gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that ablated residual expression from the endogenous OTC gene, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet.
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.|State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Lili
authors.initials: L
authors.lastname: Wang
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Bell
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Deirdre
authors.initials: D
authors.lastname: McMenamin
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Zhenning
authors.initials: Z
authors.lastname: He
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: John
authors.initials: J
authors.lastname: White
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Yu
authors.affiliation: Center for Genetic Medicine Research, Children's Research Institute, Children's National Health System, Washington, DC, USA.
authors.forename: Chenyu
authors.initials: C
authors.lastname: Xu
authors.affiliation: Center for Genetic Medicine Research, Children's Research Institute, Children's National Health System, Washington, DC, USA.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Morizono
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.|Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.
authors.forename: Kiran
authors.initials: K
authors.lastname: Musunuru
authors.affiliation: Center for Genetic Medicine Research, Children's Research Institute, Children's National Health System, Washington, DC, USA.
authors.forename: Mark L
authors.initials: ML
authors.lastname: Batshaw
authors.affiliation: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Wilson
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics; D004195:Disease Models, Animal; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D009954:Ornithine Carbamoyltransferase / Q000235:genetics* / Q000627:therapeutic use; D020163:Ornithine Carbamoyltransferase Deficiency Disease / Q000235:genetics / Q000628:therapy*; D017393:RNA Editing; D014780:Viruses / Q000235:genetics
chemical_list: D009954:Ornithine Carbamoyltransferase
references.citation: Hum Gene Ther. 2012 May;23(5):533-9
references.pmid: 22098408
references.citation: J Pediatr. 2013 Feb;162(2):324-9.e1
references.pmid: 22901741
references.citation: Gene Ther. 2013 Oct;20(10):987-96
references.pmid: 23759700
references.citation: Nat Protoc. 2013 Nov;8(11):2281-308
references.pmid: 24157548
references.citation: Blood. 2013 Nov 7;122(19):3283-7
references.pmid: 24085764
references.citation: Cell. 2014 Jun 5;157(6):1262-78
references.pmid: 24906146
references.citation: Nat Biotechnol. 2014 Jun;32(6):551-3
references.pmid: 24681508
references.citation: Mol Genet Metab. 2014 Sep-Oct;113(1-2):127-30
references.pmid: 25135652
references.citation: Nat Commun. 2014;5:5507
references.pmid: 25425480
references.citation: Science. 2014 Nov 28;346(6213):1258096
references.pmid: 25430774
references.citation: FEBS Lett. 2014 Nov 3;588(21):3954-8
references.pmid: 25241167
references.citation: Nature. 2015 Jan 15;517(7534):360-4
references.pmid: 25363772
references.citation: Nat Biotechnol. 2015 Feb;33(2):187-97
references.pmid: 25513782
references.citation: Biotechnol J. 2015 Feb;10(2):258-72
references.pmid: 25663455
references.citation: J Immunol Res. 2015;2015:397879
references.pmid: 25759840
references.citation: Nature. 2015 Apr 9;520(7546):186-91
references.pmid: 25830891
references.citation: Nat Biotechnol. 2015 Jun;33(6):584-6
references.pmid: 25985262
references.citation: Hum Gene Ther. 2015 Jul;26(7):432-42
references.pmid: 26086867
references.citation: Nature. 2015 Jul 23;523(7561):481-5
references.pmid: 26098369
references.citation: Mol Ther. 2015 Aug;23(8):1298-307
references.pmid: 26022732
references.citation: Blood. 2015 Oct 8;126(15):1777-84
references.pmid: 26297739
references.citation: Genome Biol. 2015;16:257
references.pmid: 26596280
references.citation: Pediatr Res. 2000 Dec;48(6):842-6
references.pmid: 11102556
references.citation: Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11854-9
references.pmid: 12192090
references.citation: Methods Mol Biol. 2004;246:195-9
references.pmid: 14970593
references.citation: Proc Natl Acad Sci U S A. 1989 Jun;86(11):4142-6
references.pmid: 2471197
references.citation: Hepatology. 1996 Aug;24(2):407-11
references.pmid: 8690412
references.citation: Biochem J. 1997 Mar 1;322 ( Pt 2):625-31
references.pmid: 9065786
references.citation: Nat Rev Microbiol. 2005 Nov;3(11):837-47
references.pmid: 16261169
references.citation: Mol Ther. 2006 Jul;14(1):25-33
references.pmid: 16677864
references.citation: Mol Ther. 2008 Jun;16(6):1081-8
references.pmid: 18414478
references.citation: Curr Gene Ther. 2009 Apr;9(2):104-14
references.pmid: 19355868
references.citation: Hum Gene Ther. 2010 Oct;21(10):1259-71
references.pmid: 20497038
references.citation: Mol Ther. 2011 Nov;19(11):2012-20
references.pmid: 21811248
references.citation: Gene Ther. 2012 Apr;19(4):404-10
references.pmid: 21850052