title: Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno-associated virus/piggyBac transposase gene delivery system.
abstract: Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase-deficient Spf(ash) mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data.
  
  
  CONCLUSIONS
  Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration.
authors.affiliation: Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.|University of Sydney Medical School, Sydney, New South Wales, Australia.
authors.forename: Sharon C
authors.initials: SC
authors.lastname: Cunningham
authors.affiliation: Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Siew
authors.affiliation: Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.
authors.forename: Claus V
authors.initials: CV
authors.lastname: Hallwirth
authors.affiliation: Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.
authors.forename: Christine
authors.initials: C
authors.lastname: Bolitho
authors.affiliation: Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.
authors.forename: Natsuki
authors.initials: N
authors.lastname: Sasaki
authors.affiliation: Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.|Department of Chemistry and Biomolecular Sciences, Macquarie University, Macquarie Park, New South Wales, Australia.
authors.forename: Gagan
authors.initials: G
authors.lastname: Garg
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Iacovos P
authors.initials: IP
authors.lastname: Michael
authors.affiliation: Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.
authors.forename: Nicola A
authors.initials: NA
authors.lastname: Hetherington
authors.affiliation: Biochemical Genetics, The Children's Hospital at Westmead, Westmead, Sydney, New South Wales, Australia.
authors.forename: Kevin
authors.initials: K
authors.lastname: Carpenter
authors.affiliation: Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.
authors.forename: Gustavo
authors.initials: G
authors.lastname: de Alencastro
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.|Institute of Medical Science and Department of Obstetrics & Gynaecology, University of Toronto, Toronto, Ontario, Canada.|Department of Obstetrics & Gynaecology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Andras
authors.initials: A
authors.lastname: Nagy
authors.affiliation: Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.|Discipline of Paediatrics and Child Health, The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Ian E
authors.initials: IE
authors.lastname: Alexander
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000831:Animals, Newborn; D004195:Disease Models, Animal; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000494:pharmacology*; D006801:Humans; D022124:Hyperammonemia / Q000175:diagnosis / Q000628:therapy*; D008107:Liver Diseases / Q000628:therapy; D051379:Mice; D008822:Mice, Transgenic; D012720:Severity of Illness Index; D018709:Statistics, Nonparametric; D014508:Urea / Q000378:metabolism*
chemical_list: D014508:Urea