Sharon C Cunningham1,2, Susan M Siew1, Claus V Hallwirth1, Christine Bolitho1, Natsuki Sasaki1, Gagan Garg1,3, Iacovos P Michael4, Nicola A Hetherington1, Kevin Carpenter5, Gustavo de Alencastro1, Andras Nagy4,6,7, Ian E Alexander1,8. 1. Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia. 2. University of Sydney Medical School, Sydney, New South Wales, Australia. 3. Department of Chemistry and Biomolecular Sciences, Macquarie University, Macquarie Park, New South Wales, Australia. 4. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. 5. Biochemical Genetics, The Children's Hospital at Westmead, Westmead, Sydney, New South Wales, Australia. 6. Institute of Medical Science and Department of Obstetrics & Gynaecology, University of Toronto, Toronto, Ontario, Canada. 7. Department of Obstetrics & Gynaecology, University of Toronto, Toronto, Ontario, Canada. 8. Discipline of Paediatrics and Child Health, The University of Sydney, Sydney, New South Wales, Australia.
Abstract
UNLABELLED: 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. CONCLUSION: 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.
UNLABELLED: 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. CONCLUSION: 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: Grant J Logan; Allison P Dane; Claus V Hallwirth; Christine M Smyth; Emilie E Wilkie; Anais K Amaya; Erhua Zhu; Neeta Khandekar; Samantha L Ginn; Sophia H Y Liao; Sharon C Cunningham; Natsuki Sasaki; Martí Cabanes-Creus; Patrick P L Tam; David W Russell; Leszek Lisowski; Ian E Alexander Journal: Nat Genet Date: 2017-06-19 Impact factor: 38.330
Authors: Samantha L Ginn; Anais K Amaya; Sophia H Y Liao; Erhua Zhu; Sharon C Cunningham; Michael Lee; Claus V Hallwirth; Grant J Logan; Szun S Tay; Anthony J Cesare; Hilda A Pickett; Markus Grompe; Kimberley Dilworth; Leszek Lisowski; Ian E Alexander Journal: JHEP Rep Date: 2019-12-27
Authors: Nadia Savy; David Brossier; Catherine Brunel-Guitton; Laurence Ducharme-Crevier; Geneviève Du Pont-Thibodeau; Philippe Jouvet Journal: Hepat Med Date: 2018-09-12
Authors: Que T La; Binhai Ren; Grant J Logan; Sharon C Cunningham; Neeta Khandekar; Najah T Nassif; Bronwyn A O'Brien; Ian E Alexander; Ann M Simpson Journal: Cells Date: 2020-10-02 Impact factor: 6.600