22.5 Advantages of transposon in gene delivery system:

(i) Low cost as compared to viral vectors

The extensive uses of clinical grade viral vectors are now being constrained due to introduction of transposons because they are very expensive to manufacture. Viral vectors have limitation of less GMP certified production facilities. Production of clinical Good Manufacturing Practice (cGMP) involves lot of time, standardization of cell culture conditions, testing of microbial contamination, presence of viral particles having the ability to replicate, validity of sequence and their functioning. Limited shelf life of viral stocks is also one of the major drawbacks. In contrast, cGMP grade transposon plasmids can be manufactured more quickly. Scaling up of production is easy and in a much shorter time period upgradation and certification of existing facilities can be done. For gene delivery system the use of transposons decreases both the time and cost of production.

(ii) Delivery of large and multiple transgenes

For delivering multiple transgenes, retroviral and lentiviral vectors have been successfully used but these systems can carry a limited cargo of up to 8kb which is being limited by the packaging capacity of their capsid envelope. In earlier reports it has been found out that the efficiency of sleeping beauty system has reduced beyond transposon size of 10kB. On the other hand, piggyBac system is utilized to modify primary human lymphocytes with 15 kB transposon having an initial transfection efficiency of 20% which has increased upon selection and expansion to 90%. Mobilization of large transposons like 100kb the piggyBac system is used in mouse embryonic stem (ES) cells. The significance of an increased cargo capacity is that it helps in delivering multiple transgenes to the same cell. As for example, efficient modification of human cells to express three subunit functional sodium channel using piggyBac system which helped in retaining its electro-physiological properties after 35 passages.

(iii) Less immunogenicity

In the year 1999, the death of a patient on receiving liver targeted adenoviral gene therapy for partial deficiency of ornithine transcarbamylase was due to immunogenicity. Within four days of administration of vector there was cytokine rush in the body which resulted in multiple organ failure. Various attempts have been made to reduce the immunogenicity of viral vectors by removing all the endogenous viral genes but even though such viral vectors are found to be potentially immunogenic. This has been proved by long term inflammation of rat brains which have been injected with adenoviral vectors that are replication deficient. TNF and IFN-α are inflammatory mediators which are produced when Toll-like receptor (TLR)-9 recognizes DNA with unmethylated CpG dinucleotides in the endosome. DNA-dependent activator of interferon (IFN)-regulatory factors (DAI), RNA polymerase III (Pol III), absent in melanoma 2 (AIM2), leucine-rich repeat (inFlightless I) interacting protein-1 (Lrrfip1), DExD/H box helicases (DHX9 and DHX36) and IFN-inducible protein IFI-16 are other mechanisms of innate immune sensing of naked DNA. To initiate immune response to the delivered DNA, these molecules use independent and overlapping signaling pathways.

(iv) Less tendency for oncogenic mutations:

For insertion of genes in SupT1 and Jurkat cells, human immunodeficiency virus (HIV) is preferred. Though murine leukemia virus (MLV) derived vectors are used for stable gene transfer but they give more preference to transcriptional start sites (TSS) for integration. In French X-SCID gene therapy trial, the integrations near the promoter of the LMO2 proto-oncogene have been associated with leukemia. The genome mapping of sleeping beauty transposons in mammals have showed that it is more partial to transcriptional units and upstream regulatory sequences which varies between different cell types. In primary human cells and cell lines piggyBac has no significance of integration site and has no preferred sites in chromosome too. The preference sites for integration of piggyBac are RefSeq genes, near TSS and CpG enriched motifs. This may be due to nature of the state of the cell or type of the cell. To improve the safety of gene transfer, both sleeping beauty and piggyBac are engineered for site-directed gene delivery. Till date, transposons have not been used in humans although one clinical trial has been approved.