1101. Fusion Proteins Consisting of the Sleeping Beauty Transposase and the Polydactyl Zinc Finger Protein E2C Direct Transposon Integration into a Unique Human Chromosomal Sequence
Authors: Stephen R. Yant, Yong Huang, Mark A. Kay
Journal: Molecular Therapy (2005)

The DNA transposon Sleeping Beauty (SB) can integrate efficiently into host cell genomes. Although this process makes these elements attractive vehicles for therapeutic gene delivery, the nonspecific nature of integration presents inherent hazards. To alleviate these risks, we have engineered a new class of fusion proteins comprised of a hyperactive SB transposase mutant (HSB5, 1,000% of SB10 activity) fused to either the N-terminus (SB/E2C) or C-terminus of the polydactyl zinc finger protein E2C (E2C/SB) in an attempt to direct transposon integration into predetermined chromosomal sites. Eleven different chimeric transposases containing variable-length inter-domain linkers were expressed in plasmid-transfected mammalian cells and tested for their ability to stably integrate a co-transfected neomycin-marked transposon. Despite equivalent steady-state levels in transfected cells, only fusions proteins with the E2C/SB configuration showed any significant level of integration activity, with the best chimera retaining |[sim]|5% of HSB5 activity (52% of SB10 activity). Importantly, E2C/SB-mediated integration was abrogated by single amino acid substitutions that inactivate the SB enzyme, suggesting that integration by the fusion protein was indeed SB-mediated. Remarkably, co-expressing limiting amounts of native HSB5 protein together with the fusion protein caused a strong synergistic increase in integration frequencies relative to cells expressing either protein alone. This suggests that chimeric transposases can function either alone or as mixed multimers within the tight constraints of a transposon-transposase complex. We also explored the DNA-binding capabilities of this chimera using an electrophoretic mobility shift assay. Results showed that the E2C/SB protein could bind specifically to both the 18-bp E2C recognition sequence and a canonical SB binding site, but that its affinity for the latter was markedly reduced relative to HSB5. This provides one explanation for the reduced activity of the fusion protein and suggests that the formation of mixed multimers may enhance integration activity by stabilizing the DNA-binding capabilities of the chimera. Finally, we studied target site selection by the E2C/SB fusion protein using an inter-plasmid transposition assay. We transfected HeLa cells with ampr target plasmids containing a single E2C-binding site, or mutant E2C site as a control, together with plasmids encoding the E2C/SB fusion protein and a kanamycin-marked transposon. We then prepared DNA from these cells, identified integrations by screening for a kanr/ampr phenotype in E.coli, and then compared insertion site distributions between the two groups. Sequence analysis of |[sim]|70 insertions from each group suggests that: (i) the E2C/SB fusion protein can bias integration near the E2C binding site via sequence-specific DNA binding, and (ii) that E2C/SB-mediated integrations represent true transpositions, as evidenced by the presence of flanking target site duplications. These proof-of-principle results demonstrate that E2C/SB fusion proteins offer an efficient and versatile framework for directing transposon integration into predetermined DNA sites.