Identification of the Host Interacting Factors with piggyBac Transposon for Advancing the Technology of Site-Specific Genome Targeting

Project: National Science and Technology CouncilNational Science and Technology Council Academic Grants

Project Details

Abstract

Gene therapy is a therapeutic procedure that restoring the altered encoded gene enables to perform its normal functions for maintaining the physiological homeostasis of individual. After human genome decoyed, seeking the permanent cure for diseases with genetic alterations becomes one of future trends in healthcare. Several successful clinical trials for gene therapy used to adopt virus-based strategy for therapeutic genes delivery. Yet, the raising safety issues regarding patient’s immune responses as well as the potential of endogenous gene disruption hampered the further use of virus-mediated therapeutic vector system. The development of the safe therapeutic vehicle for gene therapy has long been considered as one of the most urgent needs in the current field of gene therapy. Transposon is a mobile genetic element that engaging a “cut-and paste” mechanism to transpose within its host genomes. piggyBac transposon that originally isolated from the genome of cabbage looper moth is capable of mobilizing across phylum in mammalian genomes. It is one of the most active mobile elements among several transposon families that have been identified so far (Wu, et al., 2006). Although it possesses a high transposition activity in mammalian cells, works from our lab showed that its transposition activity displays in a host dependent manner (Meir, et al., 2013). To further improve its transposition activity, my lab had developed a series of recombinant piggyBac transposases and found that the enhancement of transposition activity is due to improving its catalytic function, but not the increment of protein production as seen in hyperactive piggyBac. Further, this pioneered works showed that the “cut” and “paste”, a two-step catalytic function of transposase, is molecularly distinguishable; i.e. our TPLGMH recombinant enhances its “paste” but not the “cut” step of its catalytic function to promote the overall transposition activity (Meir, et al., 2013). As forty-four percent of the human genome consists of the transposon-like repetitive element, the intimate interaction between a transposon and its host requires mechanisms that constrain the potential lesion imposing upon the host genome during transposition while still allow the minimal occurrence of transposition to create genome plasticity in a versatile environment during evolution. With the aforementioned results and observations, I hypothesize that there exists the taming mechanism in host for the regulatory mobilization by providing a limited permit of transposition to reach the goal of mutual benefit during genome evolution. To prove the existence of such taming mechanism, I proposed to isolate the host factors which either interact with piggyBac transposase or its TRDs. Analyzing those interacting players will reveal how the host permits transposition to happen while still maintains the integrity of its genome. Additionally, identifying those interacting factors may facilitate the establishment of a safer genome manipulation platform by enhancing its transposition activity and insertion precision. Accordingly, the third year’s proposal will focus on the development of piggyBac-mediated site-specific genome targeting by introducing the TALE technology. The HPRT locus will be used to exercise the site-specific genome targeting by taking the advantage of HAT selection after insertion in the presence or absence of candidate molecules. As a monument of non-viral vehicle for transgenesis, this proposal adopts piggyBac as a paradigm of DNA transposon to study the mutual benefits of transposon and its host during genome evolution. While exploring the host regulatory mechanism on the action of transposon, those findings can be directly applied to facilitate the development of a safer therapeutic vehicle for gene therapy.

Project IDs

Project ID:PC10308-0669
External Project ID:MOST103-2320-B182-016
StatusFinished
Effective start/end date01/08/1431/07/15

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