Episomes and Transposases-Utilities to Maintain Transgene Expression from Nonviral Vectors

Abstract

The efficient delivery and stable transgene expression are critical for applications in gene therapy. While carefully selected and engineered viral vectors allowed for remarkable clinical successes, they still bear significant safety risks. Thus, nonviral vectors are a sound alternative and avoid genotoxicity and adverse immunological reactions. Nonviral vector systems have been extensively studied and refined during the last decades. Emerging knowledge of the epigenetic regulation of replication and spatial chromatin organisation, as well as new technologies, such as Crispr/Cas, were employed to enhance the performance of different nonviral vector systems. Thus, nonviral vectors are in focus and hold some promising perspectives for future applications in gene therapy. This review addresses three prominent nonviral vector systems: the Sleeping Beauty transposase, S/MAR-based episomes, and viral plasmid replicon-based EBV vectors. Exemplarily, we review different utilities, modifications, and new concepts that were pursued to overcome limitations regarding stable transgene expression and mitotic stability. New insights into the nuclear localisation of nonviral vector molecules and the potential consequences thereof are highlighted. Finally, we discuss the remaining limitations and provide an outlook on possible future developments in nonviral vector technology.

Keywords: EBV; S/MAR; episomes; gene therapy; nonviral vectors; sleeping beauty; transposons.

Figure 1

Vector families and vector groups for gene therapy. Vector systems can be classified into families of viral and non-viral vectors. Viral vectors deliver nucleic acids to a variety of cells via transduction. Nonviral vectors are usually delivered using different transfection methods, such as lipofection or electroporation. Both viral and nonviral vectors can be grouped into integrating and nonintegrating vectors. Examples for each vector group are given without any claim of completeness. AAV, Adeno-associated Virus; HSV, Herpes Simplex Virus; S/MAR, scaffold/matrix attachment region; EBV, Eppstein-Barr virus.

Figure 2

Mechanisms of DNA replication and nuclear retention of different nonviral vector systems. (A) EBV-based replicons replicate EBNA1 and MCM2-7 dependent. The binding of EBNA1 to the DS element recruits MCM2-7, thus mediating DNA replication, while binding of EBNA1 to both the FR element and metaphase chromosomes provides a “piggy-back” mechanism for nuclear retention and segregation. (B) SB transposases are delivered to the target cells either directly as recombinant protein or as a transposes-encoding plasmid, or mRNA, along with transposon-carrying plasmids (transgene flanked by TIR sites). Once both components (transposon and transposase) are available in the cell, the SB transposase binds to the TIR, excises the transposon and mediates its integration in the cellular genome. Subsequently, the inserted transposon is replicated and segregated during the cell cycle. (C) Episomal maintenance of S/MAR-based vectors relies on the presence of active transcription running into the S/MAR sequence. Replication of S/MAR-based episomes is initiated during the early S-phase at various sites of the vector, depending on the host’s cell replication machinery. Segregation and nuclear retention rely on the interaction of the episomes with metaphase chromosomes in a “piggy-back” manner, although the proteins involved are still unknown. EBV, Eppstein-Barr virus; FR, family of repeats element; DS, dyad symmetry element; TIR, terminal inverted repeats; EBNA1, EBV nuclear antigen 1; MCM2, minichromosome maintenance complex component 2; S/MAR, scaffold/matrix attachment region.