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.
Bibliographical noteFunding Information:
We thank Nikunj Somia (Department of Genetics, Cell Biology and Development, University of Minnesota) for plasmid pIBI31, used for in vitro transcription of β-globin-stabilized luciferase-encoding messenger RNA, and the Biological Imaging and Processing Laboratory at the University of Minnesota for technical assistance. This work was supported by start-up funds from the University of Minnesota Medical School (X.W.), a grant from the Arnold and Mabel Beckman Foundation (S.C.E. and R.S.M), the Medical Scientist Training Program (K.J.W.), and by training grant T32 GM08347 from the National Institute of General Medical Sciences (A.W.). A.W. is the recipient of a University of Minnesota Doctoral Dissertation Fellowship.