Chimeric antigen receptor (CAR) T cells have tremendous promise for treatment of cancer and other conditions. Multiple CAR-T therapies have attained FDA approval in the US but a number of barriers continue to hinder their wide-scale use. One such impediment is the time and expense required to manufacture sufficient modified T cells, partly because most are produced using viral vectors like adeno-associated viruses (AAV) and lentiviruses.  Use of viral vectors can be immunogenic and produce cytotoxic effects. The viral genome may integrate randomly into the host cell genome and carry a high risk of insertional mutagenesis.  Some like to insert DNA into transcriptionally active sites, endangering cell viability and proper function.  An alternative mode of DNA insertion may change this: the Sleeping Beauty (SB) transposon system.
Transposons occur in many organisms, from plants, to insects, to vertebrates.  They are genetic elements with the ability to relocate within the genome, not unlike a train car that can unlatch itself from the cars adjacent to it and then jump into a new spot in the column of cars. They accomplish this with two functional elements: terminal inverted repeats (TIR) and a transposase gene. The transposase gene product recognizes the TIRs that flank the strand containing the transposase gene. The enzyme cuts the DNA at the TIR sites, freeing the transposon sequence, which it then reinserts at a new location.
Scientists realized this two part system could be de-coupled. They separated the TIRs from the transposon sequence they had been flanking. Instead, a gene of interest (GOI) on a plasmid can be placed between TIRs and introduced in a host cell. The transposase can enter in multiple ways, such as encoded on a plasmid of its own, on an mRNA molecule or as a protein product. One way or another, the transposase enzyme will then cut out the GOI from its plasmid and insert it into the host genome.
The Sleeping Beauty transposon system was used in humans as a clinical gene transfer tool for the first time to create CAR-T cells designed to target the SLAMF7 marker in patients with refractory multiple myeloma. The hyperactive SB100X transposase researchers used was delivered into host cells using mRNA to intentionally make transposase expression transient, decreasing the long-term risk of any unintended alteration in healthy cells. 
The SLAM7-CAR T cell Phase I/II clinical trial dubbed, CARAMBA is ongoing as a collaborative effort between six countries in Europe. 
- Field A-C, Vink C, Gabriel R, Al-Subki R, Schmidt M, Goulden N, et al. (2013) Comparison of Lentiviral and Sleeping Beauty Mediated αβ T Cell Receptor Gene Transfer. PLoS ONE 8(6): e68201. https://doi.org/10.1371/journal.pone.0068201
- Vannucci, L., Lai, M., Chiuppesi, F., et al. Viral Vectors: a look back and ahead on gene transfer technology. New Microbiologica, 36, 1-22, 2013.
- Ivics, Z., Izsvák, Z. The expanding universe of transposon technologies for gene and cell engineering. Mobile DNA 1, 25 (2010). https://doi.org/10.1186/1759-8753-1-25
- Prommersberger, S., Reiser, M., Beckmann, J. et al. CARAMBA: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleeping Beauty gene transfer to treat multiple myeloma. Gene Ther 28, 560–571 (2021). https://doi.org/10.1038/s41434-021-00254-w
- SLAM7-CAR T cell treatment of multiple myeloma patients. CARAMBA. (2022, March 14). Retrieved March 6, 2023, from https://www.caramba-cart.eu/the-project/