In Vivo Correction of Murine Tyrosinemia Type I by DNA-Mediated Transposition
| Autor: | Muhsen Al-Dhalimy, Eugenio Montini, Patrice K. Held, Milton J. Finegold, Meenakshi Noll, Markus Grompe, Mark A. Kay, Stephen R. Yant, Nicolas Morcinek |
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| Rok vydání: | 2002 |
| Předmět: |
Transposable element
Time Factors Hydrolases Transgene Biology Mice 03 medical and health sciences 0302 clinical medicine Plasmid Drug Discovery Genetics Animals Molecular Biology Transposase 030304 developmental biology Mice Knockout Recombination Genetic Pharmacology 0303 health sciences Expression vector Tyrosinemias DNA Genetic Therapy Sleeping Beauty transposon system Molecular biology Survival Rate Phenotype Liver Naked DNA 030220 oncology & carcinogenesis DNA Transposable Elements Fumarylacetoacetate hydrolase Molecular Medicine |
| Zdroj: | Molecular Therapy. 6(6):759-769 |
| ISSN: | 1525-0016 |
| DOI: | 10.1006/mthe.2002.0812 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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