Messenger RNA as a source of transposase for sleeping beauty transposon-mediated correction of hereditary tyrosinemia type I.
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.
Wilber
Andrew
A
The Arnold and Mabel Beckman Center for Transposon Research, University of Minnesota, Minneapolis, Minnesota, USA.
Wangensteen
Kirk J
KJ
Chen
Yixin
Y
Zhuo
Lijuan
L
Frandsen
Joel L
JL
Bell
Jason B
JB
Chen
Zongyu J
ZJ
Ekker
Stephen C
SC
McIvor
R Scott
RS
Wang
Xin
X
D000818:Animals; D001483:Base Sequence; D002875:Chromosomes / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D022781:Hepatocytes / Q000201:enzymology / Q000637:transplantation; D006867:Hydrolases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008107:Liver Diseases / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000628:therapy; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D019895:Transposases / Q000378:metabolism*; D020176:Tyrosinemias / Q000145:classification / Q000201:enzymology / Q000235:genetics* / Q000628:therapy
D004251:DNA Transposable Elements; D012333:RNA, Messenger; D019895:Transposases; C502724:Fahd2a protein, mouse; D006867:Hydrolases