Gene transfer into genomes of human cells by the sleeping beauty transposon system

Abstract

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.

Prolonged expression of a lysosomal enzyme in mouse liver after Sleeping Beauty transposon‐mediated gene delivery: implications for non‐viral gene therapy of mucopolysaccharidoses

“…A PCRgenerated ubiquitin C (Ub) promoter spanning bp 11-350 of the human Ub promoter (pUB6/ V5-His, Invitrogen, Grand Island, NY, USA) was inserted as a SalI-XhoI fragment into the XhoI site of pT2/BH. A XhoI-SalI fragment containing SB11 [28] and the SV40 poly(A) sequence was inserted into the XhoI at the 3′-end of the Ub promoter. The control plasmid lacked the Ub-SB11 expression cassette.…”

Transposon-based interferon gamma gene transfer overcomes limitations of episomal plasmid for immunogene therapy of glioblastoma

“…20 The pKT2/CLP vector (empty vector control) was a kind gift from Dr A Geurts (University of Minnesota). The pKT2/CLP-Luc-IRESPuro vector used facilitates expression of firefly luciferase and puromycin resistance proteins from a single mRNA and was provided by Dr Andy Wilbur (University of Minnesota).…”

“…The pT2-Luc vector has been described previously. 11 Hyperactive mutants of the SB transposase enzyme have been generated by site directed polymerase chain reaction (PCR); [20][21][22][23] in this study 'SB13', which pertains to K33A and T83A mutations in the original SB10 gene (first described by Yant et al 21 ), was expressed under the control of the PGK promoter in pPGK-SB13. A 0.5 kb DNA fragment containing the mIFN-g cDNA was excised from pORF5-mIFN-g (Invivogen, San Diego, CA) as a NcoI/SwaI fragment, and ligated into pKT2/ CLP as a NcoI/MscI fragment to generate pKT2/CLPmIFN-g.…”

Effective Expression-Independent Gene Trapping and Mutagenesis Mediated by Sleeping Beauty Transposon

“…Recently, several transposon-based vectors have been developed as alternatives for elucidation of gene functions in mouse and zebrafish (Kawakami et al, 2004;Jenkins et al, 2005;Largaespada et al, 2005;Rad et al, 2010;Clark et al, 2011). In comparison with viral vehicles, transposons can carry a large DNA fragment up to 10 kb (Geurts et al, 2003;Zayed et al, 2004;Huang et al, 2010). However, transposon-based vectors have a caveat that less than 10 copies of transposons are usually found in the genome of transgenic animals (Clark et al, 2004;Kawakami et al, 2004).…”