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 B100-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.