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In vivo correction of murine tyrosinemia type I by DNA-mediated transposition.

Abstract
Gene therapy applications of naked DNA constructs for genetic disorders have been limited because of lack of permanent transgene expression. This limitation, however, can be overcome by the Sleeping Beauty (SB) transposable element, which can achieve permanent transgene expression through genomic integration from plasmid DNA. To date, only one example of an in vivo gene therapy application of this system has been reported. In this report, we have further defined the activity of the SB transposon in vivo by analyzing the expression and integration of a fumarylacetoacetate hydrolase (FAH) transposon in FAH-deficient mice. In this model, stably corrected FAH(+) hepatocytes are clonally selected and stable integration events can therefore be quantified and characterized at the molecular level. Herein, we demonstrate that SB-transposon-transfected hepatocytes can support significant repopulation of the liver, resulting in long-lasting correction of the FAH-deficiency phenotype. A single, combined injection of an FAH-expressing transposon plasmid and a transposase expression construct resulted in stable FAH expression in approximately 1% of transfected hepatocytes. The average transposon copy number was determined to be approximately 1/diploid genome and expression was not silenced during serial transplantation. Molecular analysis indicated that high-efficiency DNA-mediated transposition into the mouse genome was strictly dependent on the expression of wild-type transposase.
AuthorsEugenio Montini, Patrice K Held, Meenakshi Noll, Nicolas Morcinek, Muhsen Al-Dhalimy, Milton Finegold, Stephen R Yant, Mark A Kay, Markus Grompe
JournalMolecular therapy : the journal of the American Society of Gene Therapy (Mol Ther) Vol. 6 Issue 6 Pg. 759-69 (Dec 2002) ISSN: 1525-0016 [Print] United States
PMID12498772 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
Chemical References
  • DNA Transposable Elements
  • DNA
  • Hydrolases
  • fumarylacetoacetase
Topics
  • Animals
  • DNA (genetics, metabolism)
  • DNA Transposable Elements (genetics)
  • Genetic Therapy (methods)
  • Hydrolases (genetics, metabolism)
  • Liver (enzymology, pathology)
  • Mice
  • Mice, Knockout
  • Phenotype
  • Recombination, Genetic (genetics)
  • Survival Rate
  • Time Factors
  • Tyrosinemias (genetics, therapy)

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