The Sleeping Beauty transposon system (SBTS) is the first successful non-viral vector for incorporation of a gene cassette into a vertebrate genome. Up until the development of this system, the major problems with non-viral gene therapy have been the intracellular breakdown of the transgene due to it being recognized as Prokaryotes and the inefficient delivery of the transgene into organ systems. The SBTS revolutionized these issues by combining the advantages of viruses and naked DNA. It consists of a transposon containing the cassette of genes to be expressed, as well as its own transposase enzyme. By transposing the cassette directly into the genome of the organism from the plasmid, sustained expression of the transgene can be attained. This can be further refined by enhancing the transposon sequences and the transposase enzymes used. SB100X is a hyperactive mammalian transposase which is roughly 100x more efficient than the typical first-generation transposase. Incorporation of this enzyme into the cassette results in even more sustained transgene expression (over one year). Additionally, transgenesis frequencies can be as high as 45% when using pronuclear injection into mouse zygotes.
The mechanism of the SBTS is similar to the Tn5 transposon system, however the enzyme and gene sequences are eukaryotic in nature as opposed to prokaryotic. The systems tranposase can act in trans as well as in cis, allowing a diverse collection of transposon structures. The transposon itself is flanked by inverted repeat sequences, which are each repeated twice in a direct fashion, designated IR/DR sequences. The internal region consists of the gene or sequence to be transposed, and could also contain the transposase gene. Alternatively, the transposase can be encoded on a separate plasmid or injected in its protein form. Yet another approach is to incorporate both the transposon and the transposase genes into a viral vector, which can target a cell or tissue of choice. The transposase protein is extremely specific in the sequences that it binds, and is able to discern its IR/DR sequences from a similar sequence by three base pairs. Once the enzyme is bound to both ends of the transposon, the IR/DR sequences are brought together and held by the transposase in a Synaptic Complex Formation (SCF). The formation of the SCF is a checkpoint ensuring proper cleavage. HMGB1 is a non-histone protein from the host which is associated with eukaryotic chromatin. It enhances the preferential binding of the transposase to the IR/DR sequences and is likely essential for SCF complex formation/stability. Transposase cleaves the DNA at the target sites, generating 3 overhangs. The enzyme then targets TA dinucleotides in the host genome as target sites for integration. The same enzymatic catalytic site which cleaved the DNA is responsible for integrating the DNA into the genome, duplicating the region of the genome in the process. Although transposase is specific for TA dinucleotides, the high frequency of these pairs in the genome indicates that the transposon undergoes fairly random integration.