1. ¹The Arnold and Mabel Beckman Center for Transposon Research, University of Minnesota, Minneapolis, Minnesota, USA
2. ²Gene Therapy Program, Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, USA
3. ³Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota, USA
4. ⁴Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA
5. ⁵Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA
6. ⁶Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, University of Minnesota, Minneapolis, Minnesota, USA
7. ⁷Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA
8. ⁸Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA

Correspondence: Xin Wang, The Stem Cell Institute, Department of Laboratory Medicine and Pathology, 2-224 McGuire Translational Research Facility, 2001 6th Street SE, University of Minnesota, Minneapolis, Minnesota 55455, USA. E-mail: wangx336@umn.edu

The first two authors contributed equally to this work.

Received 22 February 2007; Accepted 23 February 2007; Published online 17 April 2007.

Abstract

The Sleeping Beauty (SB) transposon system mediates chromosomal integration and stable gene expression when an engineered SB transposon is delivered along with transposase. One concern in the therapeutic application of the SB system is that persistent expression of transposase could result in transposon instability and genotoxicity. Here, we tested the use of transposase-encoding RNA plus transposon DNA for correction of murine fumarylacetoacetate hydrolase (FAH) deficiency. A bi-functional transposon containing both mouse FAH and firefly luciferase sequences was used to track the growth of genetically corrected liver tissue by in vivo bioluminescence imaging after delivery of DNA or RNA as a source of transposase. Supplying SB transposase in the form of RNA resulted in selective repopulation of corrected hepatocytes with stable expression of FAH and luciferase. Plasma succinylacetone and amino acid levels were normalized, suggesting normal liver metabolism of catabolized protein products. Secondary FAH-deficient animals transplanted with hepatocytes (250,000) isolated from primary treated animals survived 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC) withdrawal, gained weight consistently, and demonstrated stable expression of luciferase. We conclude that transposase-encoding messenger RNA (mRNA) can be used to mediate stable non-viral gene therapy, resulting in complete phenotypic correction, and is thus an effective source of recombinase activity for use in human gene therapy.