Comment on Huang et al, page 483

In this issue of Blood, Huang and colleagues demonstrate that the gene therapy vector system derived from the transposon system (Sleeping Beauty) can mediate stable gene transfer into unactivated primary human T lymphocytes.

Gene delivery to peripheral blood lymphocytes (PBLs) has many potential applications in the treatment of diseases such as cancer and graft-versus-host disease and in treating infectious agents. Central to successful gene therapy strategies targeting T cells is the development of an efficient gene delivery system that does not require cellular activation and is not sensitive to transcriptional silencing observed in vivo.

Huang and colleagues focus on this issue by testing a novel gene delivery strategy based on a transposon system that can deliver integrated transgenes in nonviral plasmid form. The Sleeping Beauty (SB) transposon system, initially created by Izsvak and colleagues (Ivics et al1  and Izsvak et al2 ), mediates transposon integration into TA-dinucleotide motifs throughout the genome. In an effort to demonstrate the potential of the SB system for human gene therapy, the authors demonstrate successful, stable gene delivery and expression in unactivated primary T cells. Efficient transfection was achieved when the transposase enzyme was delivered in cis or trans; however, stable gene expression was primarily observed with the trans vectors. The investigators clearly demonstrate that both CD4+ and CD8+ cells can be transfected and express the SB vectors. Analysis of T-cell clones generated from nucleofected T-cell populations revealed persistent gene expression in a small percentage of clones; however, these clones contained multiple copies of the vector. Successful integration of the SB constructs was confirmed using a splinkerette polymerase chain reaction (PCR) technique, which identified novel chromosome sequences at the integration site(s) of the clones. The study also showed the flexibility of the SB system with regard to expression of multiple gene cassettes in a series of novel constructs. The authors demonstrate the expression of multiple genes via the incorporation of the encephlomyocarditis virus internal ribosome entry site (ECMV-IRES), an 18–amino acid 2A self-cleaving oligopeptide from foot and mouth disease virus and synthetic bidirectional promoter elements. In addition, the functionality of a “suicide” gene delivered by SB was demonstrated by cytosine deaminase–mediated killing of T cells. These data represent a significant advance in the development of nonviral gene delivery systems that can mediate integration and stable gene expression.

However, there are numerous unresolved issues concerning the use of the SB system for gene therapy applications. Because mature T cells rapidly extinguish gene expression upon infusion in vivo, it is absolutely essential to analyze cell persistence and gene expression levels from SB-engineered lymphocytes in vivo. Moreover, the therapeutic potential of the SB system remains to be tested by using it to correct hereditary or acquired genetic disease. The data clearly demonstrate the integration potential associated with the transposon-based system; therefore, the risk of potential for insertional mutagenesis exists and needs to be investigated in order to fully assess the SB system. A more stringent test of the SB system, which has more relevance for gene therapy, would be to assess transposon-mediated gene delivery to hematopoietic stem cells and in vivo analysis of engraftment and differentiation. While much work remains to be completed, these studies show that Sleeping Beauty has awakened from her slumber and is ready for duty. ▪

Ivics Z, Hackett PB, Plasterk RH, Izsvak Z. Molecular reconstruction of Sleeping Beauty, a TC1-like transposon from fish, and its transposition in human cells.
Izsvak Z, Ivics Z. Sleeping Beauty transposon: biology and applications for molecular therapy.
Mol Ther.