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Sleeping Beauty Awakens to a Makeover and a Job in Genome Engineering

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In a new study, scientists from the European Molecular Biology Laboratory, the Universitätsklinikum Würzburg and the Paul-Ehrlich-Institut have designed a novel variant of the Sleeping Beauty transposase, enabling its direct use for genome modifications.

Awaking sleeping beauty

A transposon is a sequence of DNA that is able to move around and change its position within a genome. They can be used to change a cell's genetic identity by introducing genes and creating or reversing mutations, making them an efficient tool for genome engineering.

The transposon system comprises a transposase protein that binds to the end of the transposon. The transposase can be encoded by the transposon gene or it can be supplied by another source. When the transposase is supplied externally, it is known as "non-autonomous". This type of transposase is the most useful for genetic engineering, as once they are inserted, they cannot work independently and continue to re-insert themselves, providing scientists with control over the genome.

The DNA transposons that have been identified in the human genome are all non-autonomous, as, despite the fact they contain transposase genes, the genes are not functional and cannot encode a transposase that is able to mobilize the transposon.

The Sleeping Beauty transposon system comprises a Sleeping Beauty transposase that was "resurrected" from inactive copies in fish genomes in 1997. It was designed to insert specific sequences of DNA into the genomes of vertebrate animals and has been used for many applications, including gene therapy.

In a new study published in Nature Biotechnology, scientists have created a novel variant of the Sleeping Beauty transposase. This variant possesses improved biochemical properties, which the researchers suggest will enable direct use of the protein for genome modification. "The protein we developed can be delivered into mammalian cells and remains fully functional, enabling efficient and stable genome modifications in target cells on demand," explains Orsolya Barabas, group leader at EMBL Heidelberg.

Once upon a gene

The novel Sleeping Beauty transposase enables direct protein delivery and also penetrates cells autonomously, making it the first of its kind featuring this characteristic. This feature was not intentional for the variant and was only discovered through analysis of the protein. "All these features open new avenues for CAR-T cell production and other gene therapies," explains Irma Querques, Ph.D student at EMBL and a lead author of the paper.

The gene therapy space has witnessed major advances in recent years, and a number of therapies hold promise for clinical application for diseases in which treatment options are currently limited. The Sleeping Beauty system can be used to deliver efficient and precise gene engineering in several cell types, including human stem cells and T lymphocytes; the latter can be modified to produce CAR-T cells for cancer immunotherapy. "The new transposase and the genome engineering procedures we developed will find direct use in therapeutic cell engineering," highlights Michael Hudecek from the Universitätsklinikum Würzburg. He continues: "Already in this first study, we demonstrate the utility of our method for CAR-T cell production and its efficacy in a mouse model."

The Sleeping Beauty transposon system is an attractive avenue for gene therapy treatment, as it avoids the use of viral vectors which can be costly to produce and can trigger immune responses when used to insert or modify genes.

The design method used to create the novel Sleeping Beauty variant and the protocols developed by the EMBL team will be used to create similar strategies for other transposon systems. The researchers also endeavor to explore the cell penetrating property of Sleeping Beauty further and are keen to investigate whether the mechanism is transferable to other proteins. "The availability of our new Sleeping Beauty variant will also facilitate research towards understanding its molecular mechanisms, which in turn will promote the rational design of more advanced transposon tools," adds Cecilia Zuliani from EMBL; another lead author of the paper.

Further optimization of the Sleeping Beauty transposon system is required for clinical application, however, Barbas highlights, "For now, our new cell engineering procedure will lead to reduced costs and - through improved fidelity and control of the method - improved safety of medically relevant genome modifications."

Reference: Querques, I. et al. A highly soluble Sleeping Beauty transposase improves control of gene insertion. Nature Biotechnology. DOI: https://doi.org/10.1038/s41587-019-0291-z.