Preclinical and clinical advances in transposon-based gene therapy

Abstract

Transposons derived from Sleeping Beauty (SB), piggyBac (PB), or Tol2 typically require cotransfection of transposon DNA with a transposase either as an expression plasmid or mRNA. Consequently, this results in genomic integration of the potentially therapeutic gene into chromosomes of the desired target cells, and thus conferring stable expression. Non-viral transfection methods are typically preferred to deliver the transposon components into the target cells. However, these methods do not match the efficacy typically attained with viral vectors and are sometimes associated with cellular toxicity evoked by the DNA itself. In recent years, the overall transposition efficacy has gradually increased by codon optimization of the transposase, generation of hyperactive transposases, and/or introduction of specific mutations in the transposon terminal repeats. Their versatility enabled the stable genetic engineering in many different primary cell types, including stem/progenitor cells and differentiated cell types. This prompted numerous preclinical proof-of-concept studies in disease models that demonstrated the potential of DNA transposons for ex vivo and in vivo gene therapy. One of the merits of transposon systems relates to their ability to deliver relatively large therapeutic transgenes that cannot readily be accommodated in viral vectors such as full-length dystrophin cDNA. These emerging insights paved the way toward the first transposon-based phase I/II clinical trials to treat hematologic cancer and other diseases. Though encouraging results were obtained, controlled pivotal clinical trials are needed to corroborate the efficacy and safety of transposon-based therapies.

Keywords: Sleeping Beauty; Tol2; induced pluripotent stem cells; piggyBac; stem cells; transposon.

Figure 1. Molecular architecture and transposase evolution of SB and PB systems for gene delivery

(a) SB transposon is ~1.6 kb in total length and consists of two inverted repeat/direct repeats (IR/DRs) flanking DNA encoding transposase [9]. After resurrection from fish genomes, the native functional SBase (SB10) has 360 amino acids, which can be divided into DNA-binding domain (DBD) and catalytic domain. The DBDs contain two helix-turn-helix subdomains (PAI and RED subdomains) separated by GRPR-like motif [165]. The conserved Asp-Asp-Glu (DDE) trinucleotide is present in catalytic domain for DNA cleavage upon transposition [166]. SBase has undergone molecular evolution through amino acid substitutions to improve transposition efficiency for gene transfer, giving rise to more active SBase mutants such as SB11 and SB100X. The most hyperactive variant of SBase by far is ‘hySB100X’, which increases 30% of transposition rate compared with SB100X [32]. (b) PB transposon is ~2.5 kb in total size and carries two outer and inner TIRs at the end of transposon. DNA flanked by TIRs encodes 594-amino acid PB transposase (PBase) [37]. The detailed structure of PBase relatively remains elusive; however, it possesses Asp-Asp-Asp (DDD) catalytic triad for transposition [167]. Cysteine-rich motif is located at C-terminus and suggested to form plant homeodomain (PHD) finger [168]. Bipartite nuclear localization signal (NLS) is recently identified at this region [169]. Two major approaches have been employed to enhance transposition efficacy upon gene delivery: (i) codon usage optimization corresponding to mammalian hosts to promote transposase expression within the cells (i.e. mouse PBase (mPBase) and human PBase (hPBase)), and (ii) hyperactive transposase variant screening by error-prone PCR (i.e. hyperactive PBase (hyPBase)). In vivo comparative study indicates superior transposition activity of hyPBase compared with mPBase by increasing transgene expression up to 100-fold [43].