1. ¹Department of Medical & Molecular Genetics, Oregon Health and Sciences University, Portland, Oregon 97239 ⁴Department of Pediatrics, Stanford University School of Medicine, Stanford, California, 94305
2. ²Department of Pathology, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas,77030
3. ³Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305

Correspondence: Markus Grompe, Department of Molecular and Medical Genetics L103, 3181 SW Sam Jackson Park Road, Portland, OR 97201. Fax: (503) 494-6886. E-mail: grompem@ohsu.edu.

Received 3 September 2002; Accepted 28 October 2002.

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 1% of transfected hepatocytes. The average transposon copy number was determined to be 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.