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The development of vectors for genetic manipulation and gene discovery in mammalian systems

Final Report Summary - TRANSGEN (The development of vectors for genetic manipulation and gene discovery in mammalian systems)

Society welfare depends on the expansion of knowledge and technological development. Genetic engineering is one of the most powerful approaches to develop new therapeutic strategies and human-need tailored organisms. In this field, the ability to effectively introduce new genes – gene delivery techniques – and target them in the proper genetic environment is crucial.

Transposable elements (TEs) are genetic entities found in virtually all studied species. Their main trait is the ability to change their location in the genome, which is linked to an expansion of their copy numbers. TEs present a huge variability in structure, mobilization mechanism and DNA sequence. From this constellation of diversity, transposons, also known as class II TEs or cut-and-paste TE, are a very promising tool for gene delivery strategies.

In the present project, we have studied how the length of a transposon affects its ability as gene deliverer. Why are we interested in this length effect? The transposition machinery of a transposon works fine with the length of the full transposon, but there are evidences that shorter transposons are transposed with even more efficiency. Length seems to have an effect on the efficiency of transposition. Furthermore, if we bear in mind gene therapy techniques, where a non-functional gene has to be replaced with a fully functional copy, a question arises, since transposon length is shorter than most of the human genes. Thus, if transposition efficiency depends on length-dependence, it would affect negatively gene therapy success.

In our project, we have developed transposons with different lengths to uncover how the integration efficiency is affected. We have used two main approaches: in vitro and in vivo techniques. These two approaches complement each other thanks to the different backgrounds they offer for the transposition reactions. In the in vitro experiments, the main components of the reaction are mixed in a test tube with only buffer solutions. This way, we only test the molecular interactions of DNA and trasposase (the protein which catalyses transposition). On the other hand, the cellular context provides the real environment where transposition reactions occur in vivo, with all interactions with other cellular components that are not present in the in vitro tube. Our results improve our knowledge about the molecular interactions of the transposition reactions and will improve the efficiency of the integration step of the transposition reaction.

Improved transposon tools would benefit researchers in almost every academic institution in the country and around the world. The impact of our work will arise, quite simply, from the fact that improved tools make it easier to answer questions. With improved tools, more questions can be answered with a given amount of resources, but sometimes, it is also possible to answer questions that could not be asked before. Improved recombinant DNA technologies tend to be adopted very quickly because of the associated economy of effort and the large community of practitioners competing with each other. Improved tools arising from our work will therefore be made available to the wider academic community on request after publication in peer-reviewed journals.

Our particular interest is in the development of transposons as gene therapy tools. Gene therapy has been championed for the treatment of inherited conditions such as cystic fibrosis and Duchene's muscular dystrophy. It has also been explored for the treatment of cancers, tissue trauma and polygenic conditions such as diabetes, cardiovascular and neurodegenerative disease. Gene therapy offers the prospect of delivering a true medical revolution; the ability to manipulate the genome and correct genetic defects causing disease. The progress made so far is to be noted and celebrated. However, many significant hurdles remain. One of these is to assemble a gene therapy toolkit containing a range of vectors that can be used for different therapeutic purposes and targets. Viruses are currently the vector of choice and yet very few phase II or III clinical trials are being conducted. This reflects the need to explore new avenues and find new routes for efficient gene delivery. Transposon mediated therapy is one of these avenues. Successful gene therapy depends on the efficient delivery of large sections of DNA into the target cells. Building vectors able to carry sufficient genetic information to correct a complex or multi-genic disorder is no easy feat. While the cargo capacity of viral vectors is limited by the physical space available in the viral head, the amount of DNA a transposon can carry is in principal unlimited. Our research aimed at fulfilling this potential and improving the efficiency of gene delivery vectors.