Clinical Gene Transfer

Transposons ("jumping genes") are nature’s simplest gene delivery vehicles that can be harnessed as highly effective tools for versatile applications in genetic engineering, including gene therapy.

DNA transposons are genetic elements with the ability to change their positions within the genome. In nature, these elements exist as mobile units of DNA containing a transposase gene flanked by terminal inverted repeats (TIRs) that carry transposase binding sites. Importantly, it is possible to separate the two functional components of the transposon (the TIRs and the transposase) in the form of bi-component vector systems. Transposon-based vectors enable incorporation of virtually any DNA sequence of interest between the transposon TIRs and mobilization by trans-supplementing the transposase (Fig. 1). In the transposition process, the transposase enzyme mediates the excision of the element from the donor vector, followed by integration of the transposon into a chromosomal locus (Fig. 1). This feature uniquely positions transposons as non-viral gene delivery systems that unite the favorable characteristics of integrating viral vectors (i.e., stable chromosomal integration and long-lasting transgene expression) with those of non-viral delivery systems (i.e., lower immunogenicity, enhanced safety profile and reduced costs of GMP manufacture). Based on ancient, inactive transposon sequences isolated from fish genomes, an active transposon was reconstructed, and named Sleeping Beauty (SB). SB was the first transposon ever shown capable of efficient transposition in vertebrate cells, thereby enabling new avenues for genetic engineering, including gene therapy.

The advantages of SB transposon-based gene delivery include

• Permanent genomic insertion of transgene cassettes can lead to sustained and efficient transgene expression.
• In contrast to non-integrating viral vectors whose repeated in vivo administration can provoke immune responses against vector-encoded proteins, only a single administration of SB vectors is required resulting in diminished immunogenicity in vivo.
• As opposed to viral vectors that undergo a severe loss of titer beyond a certain vector size, SB vectors have no strict limitation with respect to the size of genetic cargo.
• Superior biosafety profile associated with a lack of biased integration into transcription units and transcriptional regulatory regions of genes
• In contrast to viral vectors, transposon vectors can be maintained and propagated as plasmid DNA, which makes them simple and inexpensive to manufacture, an important consideration for implementation and scale-up in clinical practice.

The Clinical Gene Transfer unit is continuously characterizing the molecular features of SB transposition in human cells, and refining tools and methods based on SB gene transfer for enhanced efficacy and safety in human gene therapy.

Previous preclinical studies contributed significant results that led to the CARAMBA clinical trial (Phase-I/IIA; EudraCT: 2019-001264-30) that investigates the feasibility, safety and anti-myeloma efficacy of autologous SLAMF7 CAR-T cells. CARAMBA is the first clinical trial in Europe that uses advanced SB technology (hyperactive SB100X transposase encoded as synthetic mRNA in conjunction with CAR transposon supplied as minicircle vectors) worldwide. Currently various active clinical trials use and examinate SB gene transfer technology.