Gene-modified cell therapy often requires stable, long-term gene expression in cells. These therapeutic genes are delivered to the nucleus of cells 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) limited payload capacity, which severely restricts the repertoire of genes that can be integrated; (2) genotoxicity arising from preferential integration of viral vectors in sites near or within active gene loci, which may negatively impact gene expression and function; (3) predisposition of silencing of genes introduced by viral vectors, presumably due to cellular immunity; and (4) immunogenicity of viral vectors. These limitations restrict clinical application of viral vectors. Additionally, viral vector-based gene therapy are expensive to manufacture given the high cost of GMP-compliant viral vectors and the stringent requirements for quality assurance.
Virus-free vector is a safer alternative due to lower immunogenicity and reduced genotoxicity compared to viral vectors. 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.
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. A commonly used two-plasmid transposon system for mediating gene integration via a simple cut-and-paste mechanism is illustrated below:
As shown in the 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 the piggyBac system for gene therapy (see Figure 1B), advancing the piggyBac transposon system for clinical applications is GenomeFrontier’s main focus. Our endeavors have resulted in the development of a potentially safer and more robust 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 with other therapeutic vectors in the context of CAR-T cell therapy arena is illustrated below (Figure 2).
Hyperactive piggyBac, currently the most advanced commercially available piggyBac system, 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 > 15 x more active than Hyperactive piggyBac and > 10,000 to 40,000 x more active when compared with the transposition efficiency of SB11 (Figure 2).
The Quantum pBac™system is detailed in Figure 3. It is 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 ensure stable gene expression. The system is safer and 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.