Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates
Key findings
Researchers developed a hyperactive Sleeping Beauty transposase (SB100X) using a phylogenetic approach and high-throughput screening of approximately 2,000 mutant clones in mammalian cells. The screen incorporated 41 hyperactive mutations from related Tc1 transposons into a combinatorial library averaging two mutations per variant. SB100X demonstrated ~100-fold increased activity compared to first-generation SB transposase and proved ~7-fold more active than codon-optimized piggyBac transposon in colony-forming assays.
SB100X achieved 35-50% stable gene transfer in hard-to-transfect human CD34+ hematopoietic stem/progenitor cells through nucleofection, with corrected transfection efficiencies indicating 70-90% of transfected cells underwent stable integration. Transplantation of gene-marked CD34+ cells into NOD-SCID γc null mice resulted in long-term engraftment (>4-5 months) with 44-73% human CD45+ cells in peripheral blood and 0.4-1.0 transposon copies per cell, confirming gene marking in both lymphoid (CD20+) and myeloid (CD33+) lineages.
Hydrodynamic injection of SB100X into adult mouse liver sustained human factor IX expression at 2,000-3,000 ng/ml for >1 year, significantly outperforming second-generation SB11 transposase (P<0.0001). In mouse transgenesis applications, pronuclear microinjection with SB100X reproducibly achieved 45% stable integration frequencies, surpassing classical methods (~10%) while avoiding concatemeric insertions that are prone to transgene silencing and recombination.