22.8 Existing hot topics and future prospects

(i) Generation of hyperactive transposon elements

In human cells similar activity level is shown by both SB100X and native piggyBac which is altogether 100 fold more than the native sleeping beauty. Two to three folds more activity is seen in hyperactive piggyBac transposase (hyPBase) than SB100X or native PB ( piggyBac ). On engineering hyperactive versions of transposase there has been increase in transposition activity. Import of amino acids from related transposases, scanning of alanine and site-directed mutagenesis are some of the strategies employed during generation of hyperactive transposon elements. ~ 100 folds higher activity is seen in SB100X transposase than the original sleeping beauty transposon. SB100X employs a high throughput screen of mutant transposes which is obtained from DNA shuffling.

Hyperactive version of piggyBac (hyPBase) transposase has been engineered with a 17-fold increase in excision and 9-fold increase in integration than the original piggyBac. It has been found that in hyPBase there are 7 amino acid substitution but none of the substitutions are present in the catalytic domain of the transposase. Foot print mutation is present in hyPBase with a frequency of <5% comparable to the wild type transposase and there is no effect on genome integrity. In SB100X there is 50% reduction even if there is an addition of 24 kDa ZFN tag which did not show any change in transposition efficiency.

(ii) Engineering of transposon systems for site-directed integration

Arbitrary insertion of transposon system has resulted in adverse conditions like leukemia. Expressions of critical genes are affected if transgenes are inserted at other genetic loci. Significance of site-directed integration: (i) improvement of gene expression, (ii) at the site of integration there is reduction of positional effects and (iii) improvement in safety. In most of the studies DNA-binding domains have been utilized to which transposase is fused to achieve site directed integration. For transposase modification piggyBac system is more suitable because the efficiency of system is not altered even by addition of domains to the tranposase. Integration of Gal4-piggyBac fusion transposase has biased the insertion near Gal4 sites in episomal plasmids and genome. A zinc finger protein (ZFP) is engineered into a chimeric transposase which is fused to native piggyBac transposase and has successfully biased the integration at genomic level. Transcription factor on DNA binding domains fused with piggyBac transposase to label transcription factor binding sites in the cells genome. Transposase have the ability to integrate on its own without targeting the machinery thereby leading to off-target integration and this is one of the major problems faced during site-directed integration. Further engineering of transposase and transposon is required to overcome such limitations.

Transposons make a promising non-viral gene delivery system due to low cost and widespread applications than viral vectors and the property for site-directed integration of gene delivery.