title: Retargeting sleeping beauty transposon insertions by engineered zinc finger DNA-binding domains.
abstract: The Sleeping Beauty (SB) transposon is a nonviral, integrating vector system with proven efficacy in preclinical animal models, and thus holds promise for future clinical applications. However, SB has a close-to-random insertion profile that could lead to genotoxic effects, thereby presenting a potential safety issue. We evaluated zinc finger (ZF) DNA-binding domains (DBDs) for their abilities to introduce a bias into SB's insertion profile. E2C, that binds a unique site in the erbB-2 gene, mediated locus-specific transposon insertions at low frequencies. A novel ZF targeting LINE1 repeats, ZF-B, showed specific binding to an 18-bp site represented by ~12,000 copies in the human genome. We mapped SB insertions using linear-amplification (LAM)-PCR and Illumina sequencing. Targeted insertions with ZF-B peaked at approximately fourfold enrichment of transposition around ZF-B binding sites yielding ~45% overall frequency of insertion into LINE1. A decrease in the ZF-B dataset with respect to transposon insertions in genes was found, suggesting that LINE1 repeats act as a sponge that "soak up" a fraction of SB insertions and thereby redirect them away from genes. Improvements in ZF technology and a careful choice of targeted genomic regions may improve the safety profile of SB for future clinical applications.
authors: Max Delbrück Center for Molecular Medicine, Berlin, Germany.
authors: Katrin
authors: K
authors: Voigt
authors: Andreas
authors: A
authors: Gogol-Döring
authors: Csaba
authors: C
authors: Miskey
authors: Wei
authors: W
authors: Chen
authors: Toni
authors: T
authors: Cathomen
authors: Zsuzsanna
authors: Z
authors: Izsvák
authors: Zoltán
authors: Z
authors: Ivics
mesh_terms: D001665:Binding Sites; D019295:Computational Biology / Q000379:methods; D004251:DNA Transposable Elements / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D016254:Mutagenesis, Insertional; D010957:Plasmids; D014162:Transfection; D019895:Transposases / Q000235:genetics* / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D019895:Transposases; C494471:sleeping beauty transposase, human