The inherent risks associated with vector insertion in gene therapy need to be carefully assessed. We analyzed the genome-wide distributions of Sleeping Beauty (SB) and piggyBac (PB) transposon insertions as well as MLV retrovirus and HIV lentivirus insertions in human CD4⁺ T cells with respect to a panel of 40 chromatin states. The distribution of SB transposon insertions displayed the least deviation from random, while the PB transposon and the MLV retrovirus showed unexpected parallels across all chromatin states. Both MLV and PB insertions are enriched at transcriptional start sites (TSSs) and co-localize with BRD4-associated sites. We demonstrate physical interaction between the PB transposase and bromodomain and extraterminal domain proteins (including BRD4), suggesting convergent evolution of a tethering mechanism that directs integrating genetic elements into TSSs. We detect unequal biases across the four systems with respect to targeting genes whose deregulation has been previously linked to serious adverse events in gene therapy clinical trials. The SB transposon has the highest theoretical chance of targeting a safe harbor locus in the human genome. The data underscore the significance of vector choice to reduce the mutagenic load on cells in clinical applications.

Clinical development of chimeric antigen receptor (CAR)-T-cell therapy has been enabled by advances in synthetic biology, genetic engineering, clinical-grade manufacturing, and complex logistics to distribute the drug product to treatment sites. A key ambition of the CARAMBA project is to provide clinical proof-of-concept for virus-free CAR gene transfer using advanced Sleeping Beauty (SB) transposon technology. SB transposition in CAR-T engineering is attractive due to the high rate of stable CAR gene transfer enabled by optimized hyperactive SB100X transposase and transposon combinations, encoded by mRNA and minicircle DNA, respectively, as preferred vector embodiments. This approach bears the potential to facilitate and expedite vector procurement, CAR-T manufacturing and distribution, and the promise to provide a safe, effective, and economically sustainable treatment. As an exemplary and novel target for SB-based CAR-T cells, the CARAMBA consortium has selected the SLAMF7 antigen in multiple myeloma. SLAMF7 CAR-T cells confer potent and consistent anti-myeloma activity in preclinical assays in vitro and in vivo. The CARAMBA clinical trial (Phase-I/IIA; EudraCT: 2019-001264-30) investigates the feasibility, safety, and anti-myeloma efficacy of autologous SLAMF7 CAR-T cells. CARAMBA is the first clinical trial with virus-free CAR-T cells in Europe, and the first clinical trial that uses advanced SB technology worldwide.

Helitron transposons capture and mobilize gene fragments in eukaryotes, but experimental evidence for their transposition is lacking in the absence of an isolated active element. Here we reconstruct Helraiser, an ancient element from the bat genome, and use this transposon as an experimental tool to unravel the mechanism of Helitron transposition. A hairpin close to the 3′-end of the transposon functions as a transposition terminator. However, the 3′-end can be bypassed by the transposase, resulting in transduction of flanking sequences to new genomic locations. Helraiser transposition generates covalently closed circular intermediates, suggestive of a replicative transposition mechanism, which provides a powerful means to disseminate captured transcriptional regulatory signals across the genome. Indeed, we document the generation of novel transcripts by Helitron promoter capture both experimentally and by transcriptome analysis in bats. Our results provide mechanistic insight into Helitron transposition, and its impact on diversification of gene function by genome shuffling.

Transposons are mobile genetic elements evolved to execute highly efficient integration of their genes into the genomes of their host cells. These natural DNA transfer vehicles have been harnessed as experimental tools for stably introducing a wide variety of foreign DNA sequences, including selectable marker genes, reporters, shRNA expression cassettes, mutagenic gene trap cassettes, and therapeutic gene constructs into the genomes of target cells in a regulated and highly efficient manner. Given that transposon components are typically supplied as naked nucleic acids (DNA and RNA) or recombinant protein, their use is simple, safe, and economically competitive. Thus, transposons enable several avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture comprising the generation of pluripotent stem cells, the production of germline-transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species and therapy of genetic disorders in humans. This review describes the molecular mechanisms involved in transposition reactions of the three most widely used transposon systems currently available (Sleeping Beauty, piggyBac, and Tol2), and discusses the various parameters and considerations pertinent to their experimental use, highlighting the state-of-the-art in transposon technology in diverse genetic applications.