Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates.
The Sleeping Beauty (SB) transposon is a promising technology platform for gene transfer in vertebrates; however, its efficiency of gene insertion can be a bottleneck in primary cell types. A large-scale genetic screen in mammalian cells yielded a hyperactive transposase (SB100X) with approximately 100-fold enhancement in efficiency when compared to the first-generation transposase. SB100X supported 35-50% stable gene transfer in human CD34(+) cells enriched in hematopoietic stem or progenitor cells. Transplantation of gene-marked CD34(+) cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution. In addition, SB100X supported sustained (>1 year) expression of physiological levels of factor IX upon transposition in the mouse liver in vivo. Finally, SB100X reproducibly resulted in 45% stable transgenesis frequencies by pronuclear microinjection into mouse zygotes. The newly developed transposase yields unprecedented stable gene transfer efficiencies following nonviral gene delivery that compare favorably to stable transduction efficiencies with integrating viral vectors and is expected to facilitate widespread applications in functional genomics and gene therapy.
Mátés
Lajos
L
Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Chuah
Marinee K L
MK
Belay
Eyayu
E
Jerchow
Boris
B
Manoj
Namitha
N
Acosta-Sanchez
Abel
A
Grzela
Dawid P
DP
Schmitt
Andrea
A
Becker
Katja
K
Matrai
Janka
J
Ma
Ling
L
Samara-Kuko
Ermira
E
Gysemans
Conny
C
Pryputniewicz
Diana
D
Miskey
Csaba
C
Fletcher
Bradley
B
VandenDriessche
Thierry
T
Ivics
Zoltán
Z
Izsvák
Zsuzsanna
Z
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D004251:DNA Transposable Elements / Q000235:genetics; D019143:Evolution, Molecular; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019895:Transposases / Q000737:chemistry / Q000235:genetics*; D014714:Vertebrates / Q000235:genetics*
D004251:DNA Transposable Elements; D019895:Transposases; C494471:sleeping beauty transposase, human