Transposons 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 (“jumping”) 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 mobilisation 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. Our group has contributed with preclinical studies using SB technology to investigate the safety and efficacy of autologous SLAMF7 CAR T cells against multiple myeloma cells. There are currently over ten active clinical trials in gene therapy making use of SB gene transfer technology. Our group is continuously characterising the molecular features of SB transposition in human cells, and actively pursuing the development and preclinical testing of novel SB transposon reagents with enhanced efficacy and safety.

Figure 1. Schematic overview of gene delivery with Sleeping Beauty transposition. The SB transposase is introduced into a cell in form of DNA (expression plasmid), mRNA or recombinant protein along with donor DNA in which the transposon to be mobilised is located. After binding within the terminal inverted repeats of the transposon (TIRs, yellow rectangles) flanking a gene of interest (GOI, green rectangle), SB transposase (blue circles) performs the excision of the transposon from the donor DNA (black strand) and integrates it into a site in the genomic target DNA (purple strand). Source: Gene Ther. 2021 Sep;28(9):560-571. doi: 10.1038/s41434-021-00254-w.

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Email: HZG6_Forschung@pei.de

Interactions between genomic parasites, such as viruses and endogenous transposable elements (TEs, mobile genetic elements known as jumping genes), and host cells can lead to their integration into the host cell genome and have negative consequences for cellular balance (homeostasis) and thus for the quality and safety of cell-based products.

With the aim of ensuring and improving the safety and efficacy of ATMPs, the research group investigates interactions between the endogenous transposable elements LINE-1 (long interspersed nuclear element-1, L1), Alu, and SVA (which are currently mobilised in the human genome) and their host cells. We seek to evaluate the risks posed by the proven activation of endogenous TEs in human pluripotent stem cells for the safety of these cells and the differentiated therapeutic cell products derived from them. Therefore, we are currently focusing on the consequences of L1-mediated retrotransposition for genomic destabilisation, expression of host genes, and the development of diseases associated with L1 activity in human pluripotent stem cells.

Although the human genome contains about 10⁶ L1 copies, which cover about 17 percent of genomic DNA, only a subset of 80-100 L1 elements encodes the complete and intact protein machinery necessary for their mobilisation and is thus capable of retrotransposition. L1 elements replicate via an RNA intermediate using a copy-and-paste mechanism. The L1-encoded protein machinery is responsible for the mobilisation of L1 elements in cis, but also for the mobilisation of non-autonomous, non-LTR human retrotransposons such as SINEs (e.g. Alu) and SVA elements, in trans (Figure 2). Any type of cellular mRNA molecules as well as, in rare cases, non-retroviral, exogenous RNA virus sequences can also be mobilised by L1-encoded proteins. This mobilisation leads to the formation of processed pseudogenes, which can impair the genomic integrity of the host cell. About 34 percent of the current human genome is generated by the activity of endogenous L1 elements. Human L1 elements are activated and mobilised in the germline, during embryonic development and the emergence of the central nervous system, and later during adult neurogenesis. The expression of endogenous L1 elements correlates with the aging process (senescence), the shortening of telomeres, stress, and DNA damage, and leads to increased L1-mediated mobilisation of TEs as observed in tumour cells. L1-mediated retrotransposition events into coding regions occur and can cause the development of diseases. Aberrant L1 overexpression can also stimulate the innate immune response, activate the immune system, and induce autoimmunity as well as inflammatory responses.

Figure 2. L1 retrotransposition cycle. A functional L1 element is transcribed and the L1 mRNA (red) is exported into the cytoplasm, translated and L1-encoded proteins (L1 ORF1p, L1 ORF2p) bind to their own mRNA (cis preference), or occasionally to Alu or SVA RNAs or cellular mRNAs (trans activity), and form ribonucleoprotein (RNP) complexes that are reimported into the cell nucleus. Subsequently, the L1 RNA (or Alu RNA, SVA RNA, or mRNA encoded by host genes) is reverse transcribed and the resulting cDNA is inserted into the genome through a mechanism called target-primed reverse transcription (TPRT). Reverse transcription typically does not progress to the 5'-end of the L1 mRNA, resulting in shortened, non-functional L1 de novo insertions. Source: Paul-Ehrlich-Institut, based on O. Weichenrieder, University of Tübingen.

Responsible for the Research Focus

Professor Dr Gerald G. Schumann
Publications
Phone: +49 6103 77 3105
Email: GeraldG.Schumann@pei.de