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Targeted gene integration in plants: vectors, mechanisms and applications for protein production

Final Report Summary - TAGIP (Targeted gene integration in plants: vectors, mechanisms and applications for protein production)

One of the limitations of current transgenics technologies in plants is that the process of DNA transformation is not precise. There is no control on the location or copy number of transgene insertion, and as a result the transgene is often silenced in unpredictable ways, immediately after transformation or there is a slow decay in the expression of the transgene throughout generations. This is problematic when trying to develop a new generation of genetically modified (GM) crops with stable transgene expression. This is even more problematic when one aims to maintain high levels of expression of the transgene, as in the case of the production of therapeutic proteins in plants (molecular pharming). There are several reports in the literature, including by members of this group, on targeted or site-specific integration of transgenes in plants. Most of these reports are still in model species and targeting efficiency rates are still too low to make it a practical, routine application. Recent advances, achieved after this project was started, have shown that GT rates can be enhanced through zinc finger nucleases (ZFNs) that can be engineered to cleave DNA at any genomic site and thus turn such DNA into hotspots for gene targeting. The ZFN technology is still in its infancy, feasibility was shown only for very few endogenous targets, it is not clear how specific is DNA break induction and its use is highly limited by intellectual property obstacles.

The goal of the TAGIP was to develop efficient targeted integration of DNA into plant genomes in order to achieve high and stable protein expression in plants. We have addressed this goal in model species (Arabidopsis and tobacco) as well as in two crops (maize and tomato). In order to achieve this goal we have developed a number of strategies. Specific goals included the development of new assays and new strategies for gene targeting; upregulation of recombination in plants to achieve gene targeting more efficiently; and protein production in plants from transgene introduced at specific sites.

We have built of a battery of constructs and transgenic plants for new gene targeting assays (based on mRFP expression in seeds); new GT systems and strategies, in Arabidopsis, tobacco, and maize, based on the use of the meganuclease I-SceI for excision of the vector from a genomic site and insertion into an engineered site also cleaved by I-SceI expression. Part of the system was to develop inducible I-SceI gene for use in maize (an achievement that lead to a patent application); preparing a series of mutants and double mutants, in genes involved in homologous or nonhomologous recombination and assessing how these affect homologous recombination, DNA repair and gene targeting; preparing a system for over-expression of bottleneck genes in a specific and controlled manner; testing the importance of Cytosine methylation on the accessibility of the target to GT. The long term goal of the project is to apply the new knowledge to applications in molecular pharming, namely the production of therapeutic proteins in plants. So far, the pharming effort has been focussed on preparing a system for site-specific DNA integration (via the Recombinase-mediated cassette exchange) and for DNA integration via homologous recombination through RAD54-mediated stimulation in tobacco and tomato.

In conclusion, the work done in TAGIP has contributed not only to the advancement of the technology of gene targeting and protein expression in plants, but also to the basic understanding of mechanisms that control DNA recombination and repair in plants, such as the role of resolvases, of non-homologous end-joining, of chromatin remodelling and of DNA methylation. We did not reach the stage of protein production from targeted integration events as originally planned, however, great progress was made towards these goals. Overall, the achievements of TAGIP have broad scientific, economical and social impact in plant biotechnology.

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