Gene-modified cell therapy often requires stable, long-term expression of therapeutic transgene(s) in cells. These therapeutic genes are delivered to a cell’s nucleus using virus or virus-free gene delivery vectors.
Viral vectors are widely used for gene therapy due to its high efficiency in gene delivery and integration with stable and long-term gene expression. However, viral vectors have several intrinsic limitations. These include: (1) the limited payload capacity which severely restrict the repertoire of genes that can be integrated; (2) genotoxicity arising from viral vector’s preference for integrating in sites near or within active gene loci, which may negatively impact the expression and/or function(s) of vital genes; (3) genes introduced by viral vectors are more prone to be silenced, presumably due to cellular immunity; and (4) safety concerns of immunogenicity of viral vectors. These limitations restrict viral vector’s clinical application. Additionally, viral vector-based gene therapies are expensive to manufacture given the high costs of GMP-compliant viral vectors and the more stringent requirements for quality assurance.
A virus-free vector obviates these shortcomings. It is safer than viral vectors due to lower immunogenicity and reduced genotoxicity. Moreover, virus-free vector has the capacity to carry larger fragments of genetic material compared to conventional viral vectors. The manufacturing costs of virus-free vectors are also markedly lower than those required for viral vector production. Despite the advantages of virus-free vectors, therapeutic genes in the form of naked DNA are short-lived and lack stable gene expression. Therefore, there remain challenges to be overcome in virus-free vector systems.
In recent years, virus-free DNA transposon has emerged as a promising vector system for gene therapy, due to its ability to overcome the challenges of gene integration. Shown below is a commonly used two-plasmid transposon system for mediating gene integration via a simple cut-and-paste mechanism:
As shown in Figure 1A above, the most common setup involves the use of a two-plasmid system – one containing the expression cassette flanked by TIRs and the other encoding the transposase. This system is capable of delivering a large payload with minimal loss of efficiency.
As one of the pioneers in the field of virus-free gene therapy, our team was the first to discover that piggyBac, a DNA transposon isolated from cabbage moths, is the most promising DNA transposon system for gene therapy as compared to other DNA transposon systems (Wu S et al., PNAS 2006, 103 (41) 15008-15013). Given the unique desirable features of piggyBac system for gene therapy (see Figure 1B ), GenomeFrontier Therapeutics, Inc. has focused on advancing the piggyBac transposon system for clinical applications. Our endeavors have resulted in a potentially safer and much more powerful piggyBac-based system, named Quantum pBac™ (Figure 3 ), which holds promise for gene therapy, particularly in CAR-T cell therapy (Meir et al., FASEB J 2013, 27 (11) 4429-4443; Meir et al., BMC Biotechnology 2011, 11:28; manuscripts in preparation). The developmental history of Quantum pBac™ in comparison to other therapeutic vectors in the context of CAR-T cell therapy arena is illustrated below (Figure 2 ).
Hyperactive piggyBac, currently the most advanced piggyBac system commercially available, and Sleeping beauty 11 (SB11) have been successfully used to develop several CAR-T cell therapies that are already in clinical trials (Figure 2 ). The transposition efficiency of Quantum pBac™ is at least 15 times more active than Hyperactive piggyBac and at least 10,000 to 40,000 times more active when compared with the transposition efficiency of SB11 (Figure 2 ).
The Quantum pBac™ system is detailed in Figure 3 . It is also a two-component system consisting of a “Donor” and a “Helper”, with the donor in a minicircle form to facilitate gene delivery, enhance gene integration, and also ensure stable gene expression. The system is safer and much more efficient as compared to the Hyperactive piggyBac system (Figure 3 ).
A direct comparison of integration efficiencies between Quantum pBac™ and Hyperactive piggyBac in human T cells shows that Quantum pBac™ is much more efficient (Figure 4 ).
These observations, along with evidence that piggyBac-based system preferentially transposes to TSCM (the most therapeutically efficacious T cell subset) make Quantum pBac™ a superior virus-free vector for application in CAR-T/TCR-T cell therapy.