Targeted Sleeping Beauty Transposition in Human Cells

Abstract

Transposons are natural gene delivery vehicles. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in the cells of vertebrates including humans. SB transposition into chromosomal DNA occurs in a fairly random manner. This is clearly not desirable in human gene therapeutic applications because there are potential genotoxic effects associated with transposon integration. In this study we set out to manipulate the selection of SB's target sites for targeted transposition into predetermined chromosomal regions. We evaluated experimental strategies based on engineered proteins composed of DNA-binding domains fused to (i) the transposase; (ii) another protein that binds to a specific DNA sequence within the transposable element; and (iii) another protein that interacts with the transposase. We demonstrated targeted transposition into endogenous matrix attachment regions (MARs) and a chromosomally integrated tetracycline response element (TRE) in cultured human cells, using targeting proteins that bind to the transposon DNA. An approach based on interactions between the transposase and a targeting protein containing the N-terminal protein interaction domain of SB was found to enable an approximately 10(7)-fold enrichment of transgene insertion at a desired locus. Our experiments provide proof-of-principle for targeted chromosomal transposition of an otherwise randomly integrating transposon. Targeted transposition can be a powerful technology for safe transgene integration in human therapeutic applications.

Development of the hybrid Sleeping Beauty-baculovirus vector for sustained gene expression and cancer therapy

“…In light of this finding, we further constructed the hybrid SB baculovirus and confirmed the efficient transduction, prolongation of transgene expression (Figures 2a and b) and the transgene persistence within the cells (Figure 2c). Since SB-mediated transposition adopts a cut-and-paste mechanism 32 and DNA-binding enzyme (for example, the flippase recombination enzyme) is able to recognize target sequences on the baculovirus and cleave the transgene off the baculovirus genome, 22 it is reasonable to envisage that the expressed SB transposase excised the genetic cargo framed by the IR/DR sequences, leading to the transgene integration as evidenced by FISH (Figure 2d).…”

Nonviral Gene Delivery with the Sleeping Beauty Transposon System

“…Target-selected SB insertion was also assessed by employing a molecular strategy based on engineering a LexA operator sequence into an SB transposon vector. Targeted transposition events into chromosomal S/MAR sequences as well as a chromosomally integrated tetracycline response element were recovered by coexpressing targeting fusion proteins containing LexA and either the SAF box, a protein domain that binds to S/MARs, or the tetracycline repressor (TetR) (Ivics et al, 2007). Presumably, these targeting fusion proteins ''sandwiched'' the transposon and target DNA sites, allowing local insertion events.…”

“…Presumably, these targeting fusion proteins ''sandwiched'' the transposon and target DNA sites, allowing local insertion events. Last, targeted SB transposition within a 2.5-kb window around a chromosomally located tetracycline response element was observed at a frequency of > 10% (Ivics et al, 2007) by coexpressing the SB transposase with a targeting fusion protein consisting of TetR and a subdomain of the SB transposase spanning the N-terminal helix-turn-helix domain (N57) that mediates protein-protein interactions between transposase subunits (Izsvák et al, 2002). Ongoing work in the authors' laboratory is dedicated to determining whether SB transposition can potentially be directed to physiologically relevant, endogenous sites in the human genome, by using both naturally occurring as well as synthetic DBDs.…”

“…1B and C), which makes them simple and inexpensive to manufacture, an important consideration for the implementation of future clinical trials. Further advantages of the SB system include its reduced immunogenicity (Yant et al, 2000), no strict limitation of the size of expression cassettes (Zayed et al, 2004), and improved safety and toxicity profiles (Ivics et al, 2007;Moldt et al, 2007;Walisko et al, 2008;VandenDriessche et al, 2009). Because transposition proceeds through a cut-and-paste mechanism that involves only DNA, transposon vectors are not prone to incorporating mutations by reverse transcription (that are generated in retroviral stocks at reasonable frequencies), and can tolerate larger and more complex transgenes.…”

Synthesis of programmable integrases

“…Synthetic zinc finger DNA-binding proteins have been generated for many sequences, using several approaches (15)(16)(17). Capitalizing on this work, researchers have incorporated synthetic zinc finger proteins into a wide variety of molecular tools (7,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).…”