The genetic engineering of the future

Today's biomedical research depends heavily on genetic engineering methods that allow scientists to remove, add or replace genes in an animal's genome. Potentially, these methods can also be used to treat disease. But most of the tools of genetic engineering have been derived from simple organisms; better ones are needed, particularly for work with humans and other vertebrates. The labs of Zsuzsanna Izsvák and Zoltán Ivics at the MDC are working to transform molecules called transposons – also known as "jumping genes" – into new tools for research and therapies. In the May 3 online issue of Nature Genetics, the researchers announce that they have made major steps toward this goal.

Genetic engineering requires molecular tools to efficiently paste new DNA sequences into chromosomes. Most of the current tools were originally found in bacteria and have been adapted for use in other species. But complex vertebrate species also hold DNA-pasting molecules – transposons. This suggested to Zsuzsanna and Zoltán that it might be possible to turn them into powerful genetic engineering toolkits that would be simpler and more effective in complex organisms.

One obstacle in the project is the fact that most transposons in vertebrates lost their functions millions of years ago. Jumping genes were likely brought into ancient animal species by parasites, including viruses. Once there, they began copying themselves and jumping, creating changes in the genome that were passed along as the organism had offspring and evolved into new species. Human DNA bears traces of at least a million such copy-paste events. But transposons are dangerous – they jump to random places, sometimes overwriting and destroying existing genes – so nearly all the copies have been deactivated by mutations.

The first task was to reactivate a transposon that Zsuzsanna and Zoltán call Sleeping Beauty. They compared about a dozen descendants of the original gene, each of which had undergone unique mutations. The comparison revealed the transposon's original spelling and allowed them to rebuild it. Since then the labs have used the same approach to reconstruct several other transposons.

But not surprisingly, the new Sleeping Beauty didn't paste itself at a very high rate in cells kept in the laboratory. It took millions of years for the original transposon to spread through an ancient vertebrate and its descendants; genetic engineers need tools that work quickly and very efficiently in single animals. And the changes that transposons make in a cell's DNA should be passed along when it divides, differentiates, and builds an organism.

How Sleeping Beauty works: The transposase (purple) docks onto inverted repeat (IR) sequences around a gene to be moved (red). The gene is cut out, then delivered to a new region of DNA, where it is inserted.

Both of these issues are addressed in the current study. One way to increase the activity of Sleeping Beauty might be to respell it, so the group began making single changes in its chemical code. Over the years, the labs of Zoltán, Zsuzsanna and others have found about 15 changes that increase the transposon's activity. But the best versions were not yet active enough. The scientists tested dozens of rewritten versions of the transposon, eventually coming up with 41 variants that pasted themselves into cells' genes at a high rate. Combining some of the spellings might produce even more active transposons, but which ones? There were an almost infinite number of combinations; trying to guess from among the millions of possiblilities would require as much luck as hitting the jackpot in a lottery. Using traditional methods, it would have been impossible to build and test all possible variants.

Postdoc Lajos Máteés and other members of the labs developed a high-throughput system to automate the process. This allowed them to create 2,000 new variants of Sleeping Beauty, integrate them into cells, and measure their activity. The experiments produced 38 highly active versions of the transposon, some of which were almost 25 times as active as the original. A study of the combinations revealed particular spellings that had the highest impact. When these were combined, the group had transposons that inserted themselves into the genome at a rate nearly 100 times higher than that of the original.

The next step was to see if the changes were long-lasting and could be inherited when cells divided. The researchers transplanted blood stem cells that had been altered through transposons into mice that were incapable of producing them; the animals now made new cells. In another experiment, the improved Sleeping Beauty was used to deliver a gene to liver cells in mice; the gene remained active for over a year.

This proves, the researchers say, that transposons can be used to deliver genes to an animal's genome at a high rate of efficiency, that the changes stay with cells as they divide and specialize, and that the effects are long-lasting. These features are crucial to genetic engineering and in modern gene therapies. So far, most such therapies have used viruses as delivery vehicles. While clinical trials of these viruses have sometimes led to complications, they are still regarded as promising tools for medical applications. But Zsuzsanna and Zoltán believe that soon, rebuilt transposons may provide a simpler, powerful method to deliver healthy genes to cells.

 - Russ Hodge

Highlight Reference:

Lajos Mátés, Marinee K L Chuah, Eyayu Belay, Boris Jerchow, Namitha Manoj, Abel Acosta-Sanchez, Dawid P Grzela, Andrea Schmitt, Katja Becker, Janka Matrai, Ling Ma, Ermira Samara-Kuko, Conny Gysemans, Diana Pryputniewicz, Csaba Miskey, Bradley Fletcher, Thierry Van den Driessche, Zoltán Ivics & Zsuzsanna Izsvák (2009). Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nature Genetics 41, 753 – 761. Published online: 3 May 2009

Link to the full article
Review article by Ivics and Izsvák on the potential of transposons for molecular medicine applications
Wikipedia article on transposons