Going non-viral: theSleeping Beautytransposon system breaks on through to the clinical side

Abstract

Molecular medicine has entered a high-tech age that provides curative treatments of complex genetic diseases through genetically engineered cellular medicinal products. Their clinical implementation requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective and economically viable manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are prevalent in ongoing pre-clinical and translational research. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here we review several recent refinements of the system, including the development of optimized transposons and hyperactive SB variants, the vectorization of transposase and transposon as mRNA and DNA minicircles (MCs) to enhance performance and facilitate vector production, as well as a detailed understanding of SB's genomic integration and biosafety features. This review also provides a perspective on the regulatory framework for clinical trials of gene delivery with SB, and illustrates the path to successful clinical implementation by using, as examples, gene therapy for age-related macular degeneration (AMD) and the engineering of chimeric antigen receptor (CAR)-modified T cells in cancer immunotherapy.

Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

“…To examine the response of the H2B-FT reporter in a situation where cell cycle speed changes concomitantly with a cell fate transition, we assessed mouse embryonic stem cells (mESCs) with either allelic expression (Iacovino et al, 2011) (details below and Figure 4D), or transposon-mediated expression (Hudecek et al, 2017) of H2B-FT. mESCs have a characteristic cell cycle of 8-10 hours that immediately lengthens upon differentiation (White, 2005). Taking advantage of this well-defined change in cell cycle rate, we induced mESC differentiation with retinoic acid (RA) (Wichterle et al, 2002) ( Figure S4F-G), which slowed the cell cycle as confirmed by EdU/DAPI staining ( Figure 4E).…”

“…mESCs were made to constitutively express H2B-FT under an EF-1α promoter using the Sleeping Beauty transposon system (Hudecek et al, 2017). H2B-FT inserts (all three variants) were cloned into a modified empty transposon (pT3) Primer sequences used for cloning are provided in Table S1.…”

Encapsulation, Visualization and Expression of Genes with Biomimetically Mineralized Zeolitic Imidazolate Framework‐8 (ZIF‐8)

“…Gene therapy holds great promise in disease treatment; however, the delivery of therapeutic genes is a severe impediment in the overall success of this approach . The efficacy of gene therapy has been greatly affected by the inadequacy of delivery vectors.…”

Genomic Analyses of SLAMF7 CAR-T Cells Manufactured by Sleeping Beauty Transposon Gene Transfer for Immunotherapy of Multiple Myeloma

“…The Sleeping Beauty (SB) transposon system [1] has been developed as a useful and promising tool for genetic engineering, including gene therapies (recently reviewed in [2][3][4][5][6][7]). The major advantage of SB gene delivery is that it combines the favorable features of viral vectors with those of naked DNA molecules, namely i) permanent insertion of transgene constructs into the genome by the transposition mechanism leads to sustained (potentially life-long) and efficient transgene expression in preclinical animal models (recently reviewed in [4][5][6]); ii) transposon vectors can be maintained and propagated as plasmid DNA, which makes vector manufacture scalable and cost-effective, and only requires S1 biological safety conditions, thereby significantly simplifying manufacture of the cell-based product. In addition, transposons can deliver larger genetic cargoes than viruses [8], and transposon-based vectors have an attractive safety profile [9][10][11][12][13].…”