Wake up the Sleeping Beauty – Transposons in gene therapy: How do they compare to other genome modifying tools?

Ewa Janosz

by Ewa Janosz

Gene therapy was believed to be a definite solution for the fast development of new therapies for  genetic diseases. However, since the first promising clinical trial in 1990 targeting ADA-SCID, only 9 approved gene therapies are available (currently 9 approved by EMA and 8 approved by FDA). We have already learned that there is a plethora of aspects to consider when developing a gene therapy.

Currently, the majority of gene therapy clinical trials use safety-optimized viral vectors and gene-editing techniques. On the other hand, there is a steadily growing number of clinical trials utilizing transposons, and it will be valuable to understand their potential in a clinical field. In this article, we will present transposons as a tool in gene therapy in reflection to other methods causing permanent changes to genomic DNA i.e., lentiviral vectors and genome-editing tools.

Transposons in gene therapy

Transposons are naturally occurring DNA sequences able to change their location in the genome. They encode for a transposase enzyme that can recognize the terminal inverted repeats (TIRs) that are flanking the transposon sequence. Transposase can cut out the flanked region and paste it in a different locus of genomic DNA. In gene therapy, for safety reasons, the transposon system is split into two vectors encoding for: i) transposase and ii) therapeutic transgene (Figure 1). Typically, the relevant sequences can be delivered to target cells ex vivo as plasmids, mRNA, or minicircles through electroporation. Until now, there have been several accepted clinical trials utilizing transposons in development of CAR-T cells and treatment of mucopolysaccharidosis (e.g. NCT04284254, a Phase 1/2 study in patients with Hurler syndrome).

Figure 1:   Representation of the transposition mechanism


Figure 1:   Representation of the transposition mechanism

How do transposons compare to other genome modifying tools?


As transposons are a non-viral transgene delivery tool, electroporation is the most common method used for cell transfection. It is a stressful treatment for cells and is associated with significant cell mortality. However, recent developments led to an improvement in post-transfection survival of hematopoietic stem and progenitor cells when using minicircles or mRNA encoding for the relevant sequences, instead of plasmids (Holstein et al., 2018). Furthermore, electroporation promises advantages in terms of minimalizing immunogenicity by bypassing the endosomal compartment (endosomal escape) (Rosi et al., 2006; Sahay et al., 2010) and lowering cytokine signaling and inflammation (Zhou et al., 2007).

Similarly, CRISPR/Cas9 or TALEN is often physically delivered to target cells through electroporation in ex vivo editing approaches. In currently explored in vivo CRISPR/Cas9 genome editing systems, adeno-associated viral vectors (AAV) are often used as a delivery method. Most prominent advantages of the AAV vectors in this context are their safety profile that was proven in multiple clinical trials, and mostly episomal persistence in transduced cells (Ates et al., 2020). On another hand, retro-/lentiviral vectors can actively transduce and enter the cell and the nucleus to integrate the transgene to the genome.

Transgene size

Another important aspect to consider when a therapeutic transgene needs to be integrated into the genome is the transgene size. In the case of transposons, there is almost unlimited insert size potential, as PiggyBac and Sleeping Beauty transposons were shown to integrate as much as a 200 kb long bacterial artificial chromosome (Li et al., 2011; Rostovskaya et al., 2012). Although a reduction in transposition efficiency is observed with increased transgene size, it potentially gives the opportunity for transfer of large transgenes, including the largest human gene encoding for dystrophin.

The transgene bearing capacity of lentiviral vectors at approximately 8 kb is often a limiting factor in designing an optimal therapeutic transgene. In addition, genome-editing techniques, like TALEN and CRISPR/Cas9, for the targeted insertion of a delivered transgene are still in development phases, with intensely studied improvements in this context. Hence, currently genome-editing tools are primarily used in gene therapy when a disruption of a genomic sequence might have a therapeutic effect, e.g., knock-out of the CCR5 gene might prevent the entry of HIV into T cells (Xu et al., 2017).

Safety- integration profile

Genome modifying methods like CRISPR/Cas9 and TALEN have the great advantage of targeted activity in a sequence of choice within the genome. However, the other tools considered in this article have more random activity. Lentiviral vectors display a random integration profile with a preference for transcriptionally active genes, which raises safety concerns in terms of insertional mutagenesis. Historically, γ-retroviral vectors were associated with development of leukemia in several patients due to insertion in proto-oncogenes (Hacein-Bey-Abina et al., 2003). However, recent improvements in lenti- and retroviral vector design, like self-inactivating configuration that abolishes the internal viral promoter, are considered as significant improvements in terms of safety.

In contrast to lentiviral vectors, transposons, and in particular the Sleeping Beauty system, present a safer, more random integration pattern, as it does not demonstrate preferences to insert the transgene in the active genes or transcriptional start sites. This results in lower probability of integration in oncogenic genes and lowers chances for insertional mutagenesis. This might explain the choice of the Sleeping Beauty over other transposons, like PiggyBac, in the majority of ongoing clinical trials.

Manufacturing cost

Gene therapies are often the only available treatment for patients. The lentiviral-based gene therapies that are currently available are also the most expensive drugs in the market, with the price tags reaching millions. This is in part due to a high cost of lentiviral vector production. In constrast, DNA/RNA-based transposon systems promise less required production steps and as a result can offer cheaper manufacturing price and more affordable therapy.

Table 1:  Comparison of genome modifying tools for gene therapy.

Table 1:  Comparison of genome modifying tools for gene therapy.


The continued research and development of transposons currently extends the available gene therapy toolkit by offering more random transgene integration and a possibility to deliver large transgenes. The associated low production cost may be another reason for the increasing uptake of transposons as more affordable alternative to e.g. lenti- and retroviral vector-based therapies. We observe an increase in translational activity and clinical development, with transposons being used ex vivo, e.g. on hematopoietic cells (Magnani et al., 2020) similarly to lentiviral vectors.

Transposon based therapeutics represent an emerging and highly promising new platform for biotherapeutics. Due to the lack of specific regulatory guidance and with many potential obstacles for drug development, however, transposons remain an especially challenging technology. With many years of experience with innovative and emerging product classes, our team of regulatory experts in the field of CMC, nonclinical, and clinical development can support you in overcoming these hurdles and challenges and in bringing your gene therapy products to the clinic and market. Contact us to learn how we can guide you to boost your product to success.


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