Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

Abstract

DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system.

Nonviral Gene Therapy—The Challenge of Mobilizing DNA

“…Using such helper-independent transposon-transposase vectors (HITT, later most often referred to as 'cis' vectors), optimized DNA transposition in mouse liver was achieved and a haemophilia B mouse model treated (Mikkelsen et al 2003). To further improve the safety profile and the efficacy of the SB system, the SB transposon vector technology was recently combined with the minicircle DNA technology (Sharma et al 2013). By exploiting minicircles as donors for SB transposons, efficient transposition and, at low transposase dosages, stable transfection rates were achieved that were improved relative to the standard plasmid donor.…”

Transposons: Moving Forward from Preclinical Studies to Clinical Trials

“…Increasing the overall transposition efficiency by using hyperactive SB100X, mini-circle plasmids or combinations thereof may also require a less prolonged in vitro enrichment period of the transposon-modified CAR T cells. 126,177,178 Even mini-circle plasmids entirely devoid of antibiotic resistance genes can potentially be used. 177 Alternatively, co-transfection of the SB transposase construct with two distinct transposons, one encoding CAR and the other mIL15, can potentially shorten the in vitro expansion period.…”

Sleeping Beauty transposon vectors for therapeutic applications: advances and challenges

“…Reducing the size of the DNA, and using supercoil DNA proved to be advantageous modifications in the delivery protocol [76,77]. Furthermore, the use of conventional plasmids as vectors that are propagated and isolated from bacteria raises a safety concern and a roadblock for broad clinical applications.…”

“…They are produced by the inclusion of site-specific intramolecular recombination motifs between the GOI and bacterial backbone in the parental plasmid. SB transposon and transposase minicircle constructs have been examined and optimized for safety and efficacy in various cell types [77], including primary T cells [76]. A minicircles-based SB system has been also used for efficient germline transgenesis [81].…”