Latest Advances for the Sleeping Beauty Transposon System: 23 Years of Insomnia but Prettier than Ever

Abstract

The Sleeping Beauty transposon system is a nonviral DNA transfer tool capable of efficiently mediating transposition-based, stable integration of DNA sequences of choice into eukaryotic genomes. Continuous refinements of the system, including the emergence of hyperactive transposase mutants and novel approaches in vectorology, greatly improve upon transposition efficiency rivaling viral-vector-based methods for stable gene insertion. Current developments, such as reversible transgenesis and proof-of-concept RNA-guided transposition, further expand on possible applications in the future. In addition, innate advantages such as lack of preferential integration into genes reduce insertional mutagenesis-related safety concerns while comparably low manufacturing costs enable widespread implementation. Accordingly, the system is recognized as a powerful and versatile tool for genetic engineering and is playing a central role in an ever-expanding number of gene and cell therapy clinical trials with the potential to become a key technology to meet the growing demand for advanced therapy medicinal products.

Contemporary Transposon Tools: A Review and Guide through Mechanisms and Applications of Sleeping Beauty, piggyBac and Tol2 for Genome Engineering

“…It was awakened from its sleep by Ivics et al in 1997 [ 21 ] via the construction of a consensus sequence from eight different fish species by elimination of the inactivating mutations. Following reconstruction, SB was also found capable of mediating transposition in a variety of vertebrates including mice and humans [ 21 , 22 ], and hence, it progressively stood up as a practical tool for genome engineering with applications from functional genomics [ 23 ] to gene and cell therapy [ 24 ].…”

“…SB supports a full spectrum of genetic engineering applications/methods (reviewed in [ 50 ]), including the generation of transgenic cell lines, induced pluripotent stem cell (iPSC) reprogramming [ 51 , 52 , 53 , 54 , 55 , 56 ], phenotype-driven insertional mutagenesis screens in the area of cancer biology (reviewed in [ 23 , 57 , 58 ]), germline gene transfer in experimental animals [ 59 , 60 , 61 , 62 , 63 , 64 , 65 ], and somatic gene therapy both ex vivo and in vivo (reviewed in [ 24 , 44 , 50 , 66 , 67 , 68 , 69 , 70 , 71 ]), which will be discussed in the following sections of this review.…”

“…Transposon systems, as outlined in this review, hold promising potential in the field of gene therapy due to their efficient, safe, and relatively simple way of shuttling transgenes into a wide range of clinically relevant cell types. SB and PB transposon systems have been successfully used in multiple preclinical studies tackling rare genetic disorders, including tyrosinemia and diabetes type I, familial hypercholesterolemia, hereditary hyperbilirubinemias, cystic fibrosis, mucopolysaccharidosis, hemophilia A and B, sickle cell disease, epidermolysis bullosa, Duchenne muscular dystrophy (DMD), and Limb-Girdle muscular dystrophies (LGMD) [ 24 , 146 , 190 , 193 , 194 ]. For these diseases, the treatment typically consists of simple complementation of the defective gene, with transposons being mediators of long-term treatment efficacy and potential curative outcome after a single dose.…”

“…However, transposon systems represent a viable alternative to mediate gene transfer, and have been continuously tested in preclinical CAR-T cell therapy settings. SB pioneered applications with the well-characterized CD19-specific CAR-T cells (reviewed in [ 24 , 206 ]), and was joined by PB [ 206 , 207 , 208 ] and Tol2 [ 113 ] to demonstrate suppression of B cell lymphoma progression both in vitro and in vivo. The most recent developments include CAR-T cell engineering for alternative targets such as the granulocyte-macrophage colony-stimulating factor receptor (hGMR or CD116) for hematological malignancies [ 209 ], the epidermal growth factor receptor (EGFR) as a target for non-small-cell lung carcinoma [ 210 ], glypican-3 and EGFRvIII targeting hepatocellular carcinoma [ 211 , 212 ] and membrane-proximal mesothelin (MSLN) epitope-targeting against MSLN-positive solid tumors [ 213 ].…”

“…Cytokine-induced killer (CIK) cells, effector lymphocytes displaying a mixed T and NK phenotype with non-HLA-restricted cytotoxicity and minimal alloreactivity, have also been generated with SB to target CD19, CD123, and CD33 [ 216 , 217 ]. Finally, SB was shown successful for T cell receptor (TCR)-T cell engineering [ 24 , 218 ], a strategy that enables to broaden the spectrum of targets as the MHC-TCR interaction also permits recognition of epitopes from intracellular proteins.…”