DNA based transposon vectors offer a mechanism for non-viral gene delivery into mammalian and human cells. These vectors work via a cut-and-paste mechanim whereby transposon DNA containing a transgene(s) of interest is integrated into chromosomal DNA by a transposase enzyme. The first DNA based transposon system which worked efficienty in human cells was
2. Transposons as gene delivery systems
Transposons or mobile genetic elements were first described by Barbara McClintock as “jumping genes” responsible for mosaicism in maize . Transposons are found in the genome of all eukaryotes and in humans at least 45% of the genome is derived from such elements . Transposons active in eukaryotes can work either by a “copy and paste” (Class I) or “cut and paste” (Class II) mechanism (Figure 1).
In the “copy and paste” mechanism, the transposon first makes a copy of itself via an RNA intermediate (hence also known as retrotransposons).Class II DNA-transposons work by a “cut and paste” mechanism in which the transposon is excised by the transposase upon expression and then relocates to a new locus by creating double strand breaks
In “cis” delivery the transposase is carried by the same plasmid backbone as the transposn. In “trans” delivery it is delivered by a separate circular plasmid. For gene therapy purposes transposase and transposon are delivered either in “cis” or in “trans” (Figure 2). In “cis” delivery the transposase is carried on the same vector backbone as the transposon carrying the gene of interest (GOI). In the “trans” configuration, the transposase is delivered by a separate non integrating plasmid. The “cis” configuration has been shown to improve transposition efficiency , but there is a question of whether the linearized backbone carrying the transposase may also get integrated and lead to residual transposase expression. A comparison of the properties of
3. Advantages of transposon as gene delivery system
3.1. Lower cost compared to viral vectors
In spite of viral vectors having been successfully used in gene therapy clinical trials (e.g. generation of clinical grade T cells for immunotherapy , their use in extensive gene therapy regimens is constrained. Clinical grade viral vectors are very expensive to manufacture given the stringent regulatory oversight and limited number of GMP certified production facilities. A batch of clinical grade retroviral supernatant for treating patients costs between $400,000 to $500,000 (personal communciation, GMP facility director, Baylor College of Medicine). The production of clinical GMP (cGMP) grade viral supernatant is extremely time intensive as, in addition to optimization of culture conditions, the supernatant needs extensive testing for microbial contamination, presence of replication competent viral particles as well as validation of sequence and functionality. The entire production run and associated testing may require up to six months. These viral stocks also have limited shelf life. Upon release the desired cell type is transduced, selected and expanded which is then followed by quality assurance checks. This also requires extensive training of the personnel involved in production and testing and scaling up production as would be required for future gene therapy regimens will not be economical. In contrast, cGMP grade transposon plasmids can be manufactured more quickly. The production can be scaled up quickly and existing facilities can be upgraded and certified in a shorter time frame. The cost of manufacturing and release of cGMP grade plasmid DNA is between $20,000 and $ 40,000 . The use of transposons drastically reduces both the time and cost of production of the gene delivery system. In the first clinical trial approved by the FDA for infusion of autologous
|~10 kb||>100 kb|
|Insertion site mutated upon excision||No “foot print” mutation|
|SB100X (most active SB version)||hyPBase|
|50% or more reduction in efficacy||No apparent reduction in efficiency|
|More random||Slight increased preference for genes and TSS|
3.2. Delivery of large and multiple transgenes
Although retroviral and lentiviral vectors have been successfully used for delivering multiple transgenes, they are limited by their cargo capacity[11,12]. Both these vector systems can carry a limited cargo of up to 8kb which is limited by the packaging capacity of their capsid envelop . Early reports demontrated the
3.3. Less immunogenicity
One of the major concerns for viral gene delivery system is the associated immunogenicity as evidenced by the death of a patient receiving liver targeted adenoviral gene therapy for partial ornithine transcarbamylase deficiency in 1999 .The systemic delivery of the viral particles initiated a cytokine storm leading to multiple organ failure within four days of administration of the vector . Attempts have been made to reduce the immunogenicity of viral vectors by stripping them of all endogenous viral genes (‘gutted’ or ‘helper-dependent’ vectors) , but even the use of modified viral delivery systems are potentially immunogenic as evidenced by long term inflammation of rat brains injected with replication deficient adenoviral vectors .
Transposons are circular plasmid DNA molecules and do not contain a viral shell or viral antigens. The host response to non-viral vectors has not been well characterized. Toll-like receptor (TLR)-9 is known to recognize DNA with unmethylated CpG dinucleotides in the endosomewhich can lead to signalling via MyD88 and production of inflammatory mediators such as TNF and IFN-α . Other mechanisms of innate immune sensing of naked DNA include DNA-dependent activator of interferon (IFN)-regulatory factors (DAI) (also called Z-DNA-binding protein 1, ZBP1), RNA polymerase III (Pol III), absent in melanoma 2 (AIM2), leucine-rich repeat (in Flightless I) interacting protein-1 (Lrrfip1), DExD/H box helicases (DHX9 and DHX36), and most recently, the IFN-inducible protein IFI16 . These molecules use independent and sometimes overlapping signalling pathways to elicit immune response to delivered DNA. Nonetheless, much remains to be discovered about host immune response to delivered DNA and how to overcome such an obstacle for effective gene therapy.
3.4. Less propensity for oncogenic mutations
Human immunodeficiency virus (HIV) has been shown to prefer genes for integration in SupT1 and Jurkat cells . Murine leukemia virus (MLV) derived vectors have been used for stable gene transfer for therapy but they have been shown to prefer transcriptional start sites (TSS) for integration . Integrations near the promoter of the LMO2 proto-oncogene has been associated with leukemia in the French X-SCID gene therapy trial . The genome wide mapping of
4. Challenges of transposon as gene delivery system
Given the promise of transposons as gene delivery vehicle, it suffers from certain challenges e.g. reduced delivery, random integration profile and silencing of the integrated transgene.
4.1. Low delivery efficiency
Transposon systems are carried by naked DNA plasmids and there efficiency is limited to the efficiency of getting the plasmid into to the cell by chemical or physical means. Certain primary cells and cell lines are easy to transfect (e.g. HEK293, HeLa, Hepatocytes) and transposons have high transposition efficiency in these cells. But other clinically relevant cells (e.g. primary lymphocytes) are difficult to transfect. Often the method used for transfection (e.g. nucleofection and electroporation) is toxic to the cells and leads to excessive cell death thus reducing the efficiency of stable transfection. Efforts are on to circumvent these difficulties by developing novel delivery methods e.g. cell-penetrating peptides (CPP) –
4.2. Random integration profile
Transposons as described above have uncontrolled or relatively random integration preference with regards to genomic elements. This leaves the transposed transgene open to influence of the neighboring genomic region. Additional, uncontrolled or not site-directed integration increases the risk for possible genotoxicity.
4.3. Silencing of the integrated transgene
Gene silencing has been observed when using
5.1. Genetic modification of human T lymphocytes
Peripheral blood and umbilical cord T cells have been extensively modified with both viral and non-viral gene delivery systems for immunotherapeutic purposes . This therapeutic avenue has been successfully used for the treatment of viral infections and Epstein Barr virus (EBV) associated lymphoma post autologous bone marrow transplantation [46,47]. They also hold promise for treatment of other cancers [48-50]. But the use of of viral vectors for the generation of clinical grade T cells is expensive, time intensive and not free of risks. Non-viral gene delivery systems, including DNA transposons, are being increasingly explored as an alternative strategy.
A schematic of how primary human T lymphocytes can be gene modified with transposons is shown in Figure 3. The
5.2. Generation of induced pluripotent stem cells
Induced pluripotent stem cells (iPSCs) generated from a patient’s own differentiated somatic cells holds promise for regenerative medicine. Early successful attempts involved delivery of defined reprogramming factors using retroviral vectors [11,53]. Unfortunately 20% of the chimeric offspring obtained from germline transmission of retrovirally reprogrammed clones developed tumors due to reactivation of the c-myc oncogene . In addition, ectopic expression of the reprogramming factor(s) has been linked to tumors and skin dysplasia [55-56]. One way to circumvent the use of viral delivery systems is to deliver the programming factors as recombinant proteins  or by repeated plasmid transfections , both of which have proven to be extremely slow and inefficient. The higher gene delivery efficiency of transposons together with their ability of being excised from the cells post reprogramming and differentiation make them an attractive choice for generating iPSCs.
Somatic cells have been transfected with
5.3. Genetic modification of stem cells
Transposons have been used for genetic modification of human embryonic stem cells . More recently, transposons have been used to insert bacterial artificial chromosomes (BACs) in human ES cells . Both
6. Current hot topics and future directions
6.1. Generation of hyperactive transposon elements
SB100X and native
Efficiency of transposition is perceived as a bottleneck to efficient gene delivery. Attempts to engineer hyperactive versions of transposase have resulted in versions with increasing transposition activity. Strategies employed include import of amino acids from related transposases , alanine scanning  and site directed mutagenesis . The construction of the SB100X transposase with ~100 folds higher activity than the original
6.2. Engineering transposon systems for site-directed integration
Random integration of transgene during delivery have resulted in adverse events including leukemia [25,72]. Integration of transgenes at other genetic loci may also affect expression of critical genes. Engineering transposon systems for site-directed integration would allow transgene delivery to sites in the genome resulting in improved gene expression, reduced positional effects at the site of integration, and improved safety. Most studies have utilized fusion of DNA-binding domains to the transposase to achieve site directed integration, beginning with the engineering of the
Transposon systems are well suited for
SS is supported in part by the Howard Hughes Medical Institute Med-Into-Grad Scholar grant to TBMM Program. MHW is supported in part by NIH R01 DK093660.
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